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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 semi-preparative scale, and the synthesized disaccharides were purified. The maximum yield obtained for LNB was 10.7 ± 0.2 g/l and for GNB was 10.8 ± 0.3 g/l. NMR spectroscopy confirmed the molecular structures of both carbohydrates and the absence of reaction byproducts, which also supports that GnbG is specific for β1,3-glycosidic linkages. The purified sugars were subsequently tested for their potential prebiotic properties using Lactobacillus species. The results showed that LNB and GNB were fermented by the tested strains of L. casei, Lactobacillus rhamnosus (except L. rhamnosus strain ATCC 53103), Lactobacillus zeae, Lactobacillus gasseri, and Lactobacillus johnsonii. DNA hybridization experiments suggested that the metabolism of those disaccharides in 9 out of 10 L. casei strains, all L. rhamnosus strains and all L. zeae strains tested relies upon a phospho-β-galactosidase homologous to GnbG. The results presented here support the putative role of human milk oligosaccharides for selective enrichment of beneficial intestinal microbiota in breast-fed infants. [ABSTRACT FROM AUTHOR]

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, demonstrating that BnaG is involved in its metabolism. Interestingly, whole cells of a bnaG mutant were totally devoid of β-N-acetylglucosaminidase activity, suggesting that BnaG is an extracellular wall-attached enzyme. In addition to hydrolyzing lacto-N-triose into N-acetylglucosamine and lactose, the purified BnaG enzyme also catalyzed the hydrolysis of 3'-N-acetylglucosaminyl-mannose and 3'-N-acetylgalactosaminyl-galactose. L. casei can be cultured in the presence of 3'-N-acetylglucosaminyl-mannose as a carbon source, but, curiously, the bnaG mutant strain was not impaired in its utilization. These results indicate that the assimilation of 3'-N-acetylglucosaminyl-mannose is independent of BnaG. Enzyme activity and growth analysis with a manA-knockout mutant showed that ManA is involved in the utilization of the mannose moiety of 3'-N-acetylglucosaminyl-mannose. Here we describe the physiological role of a β-N-acetylglucosaminidase in lactobacilli, and it supports the metabolic adaptation of L. casei to the N-acetylglucosaminide-rich gut niche. [ABSTRACT FROM AUTHOR]

The probiotic L actobacillus casei catabolizes galacto- N-biose ( GNB) and lacto- N-biose ( LNB) by using a transport system and metabolic routes different from those of B ifidobacterium. L . casei contains a gene cluster, gnbREFGBCDA, involved in the metabolism of GNB, LNB and also N-acetylgalactosamine. Inactivation of gnbC ( EIIC) or ptsI (Enzyme I) of the phosphoenolpyruvate : sugar phosphotransferase system ( PTS) prevented the growth on those three carbohydrates, indicating that they are transported and phosphorylated by the same PTSGnb. Enzyme activities and growth analysis with knockout mutants showed that GnbG (phospho-β-galactosidase) hydrolyses both disaccharides. However, GnbF ( N-acetylgalactosamine- 6P deacetylase) and GnbE (galactosamine- 6P isomerase/deaminase) are involved in GNB but not in LNB fermentation. The utilization of LNB depends on nagA ( N-acetylglucosamine- 6P deacetylase), showing that the N-acetylhexosamine moieties of GNB and LNB follow different catabolic routes. A lacAB mutant (galactose- 6P isomerase) was impaired in GNB and LNB utilization, indicating that their galactose moiety is channelled through the tagatose- 6P pathway. Transcriptional analysis showed that the gnb operon is regulated by substrate-specific induction mediated by the transcriptional repressor GnbR, which binds to a 26 bp DNA region containing inverted repeats exhibiting a 2T/2A conserved core. The data represent the first characterization of novel metabolic pathways for human milk oligosaccharides and glycoconjugate structures in Firmicutes. [ABSTRACT FROM AUTHOR]

AlfB and AlfC α-L-fucosidases from Lactobacillus casei were used in transglycosylation reactions, and they showed high efficiency in synthesizing fucosyldisaccharides. AlfB and AlfC activities exclusively produced fucosyl-α-1,3-N-acetylglucosamine and fucosyl-α-1,6-N-acetylglucosamine, respectively. The reaction kinetics showed that AlfB can convert 23% p-nitrophenyl-α-L-fucopyranoside into fucosyl-α-1,3-N-acetylglucosamine and AlfC at up to 56% into fucosyl-α-1,6-N-acetylglucosamine. [ABSTRACT FROM AUTHOR]

Goodpasture disease is an autoimmune disorder mediated by circulating autoantibodies against the noncollagenous-1 (NC1) domain of the α3 chain of type IV collagen (α3(IV)NC1). The structure of Goodpasture epitope(s) has been previously mapped into two main binding regions (EA and EB) of the α3(IV)NC1 domain using a residue mutation approach on the highly related α1(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 unrecognized within and outside of the EA or EB regions. Residue involvement in pathogenic reactivity was confirmed by site-directed mutagenesis on a more divergent α2(IV)NC1 molecule. From a mutant (M1) of the α2(IV)NC1 molecule, harboring residues previously identified as belonging to the Goodpasture epitope, additional chimeras were generated on the bases of phage display findings. All these mutants were shown to display higher reactivity with circulating Goodpasture autoantibodies than the M1 mutant. Thus, our results more precisely define Goodpasture epitope determinants and open new avenues to delineate comprehensive autoantibody-blocking agents for therapeutics. [ABSTRACT FROM AUTHOR]

Sortases are a class of enzymes that anchor surface proteins to the cell wall of Gram-positive bacteria. Lactobacillus casei BL23 harbors four sortase genes, two belonging to class A (srtA1 and srtA2) and two belonging to class C (srtC1 and srtC2). Class C sortases were clustered with genes encoding their putative substrates that were homologous to the SpaEFG and SpaCBA proteins that encode mucus adhesive pili in Lactobacillus rhamnosus GG. Twenty-three genes encoding putative sortase substrates were identified in the L. casei BL23 genome with unknown (35%), enzymatic (30%), or adhesion-related (35%) functions. Strains disrupted in srtA1, srtA2, srtC1, and srtC2 and an srtAl srtA2 double mutant were constructed. The transcription of all four sortase encoding genes was detected, but only the mutation of srtA1 resulted in a decrease in bacterial surface hydrophobicity. The Is-N- acetyl-glucosaminidase and cell wall proteinase activities of whole cells diminished in the srtAl mutant and, to a greater extent, in the srtAl srtA2 double mutant. Cell wall anchoring of the staphylococcal NucA reporter protein fused to a cell wall sorting sequence was also affected in the srtA mutants, and the percentages of adhesion to Caco-2 and HT-29 intestinal epithelial cells were reduced for the srtA1 srtA.2 strain. Mutations in srtC1 or srtC2 result in an undetectable phenotype. Together, these results suggest that SrtAi is the housekeeping sortase in L casci BL23 and SrtA2 would carry out redundant or complementary func- tions that become evident when SrtA1 activity is absent. [ABSTRACT FROM AUTHOR]

We have previously characterized from Lactobacillus casei BL23 three a-L-fucosidases, AlfA, AlfB, and AlfC, which hydrolyze in vitro natural fucosyl-oligosaccharides. In this work, we have shown that L. casei is able to grow in the presence of fucosyl-α-1,3- N-acetylglucosamine (Fuc-α-1,3-GlcNAc) as a carbon source. Interestingly, L. casei excretes the L-fucose moiety during growth on Fuc-a-1,3-GlcNAc, indicating that only the N-acetylglucosamine moiety is being metabolized. Analysis of the genomic sequence of L. casei BL23 shows that downstream from alfB, which encodes the a-L-fucosidase AlfB, a gene, alfR, that encodes a transcriptional regulator is present. Divergently from alfB, three genes, alfEFG, that encode proteins with homology to the enzyme IIAB (EIIAB), EIIC, and EIID components of a mannose-class phosphoenolpyruvate:sugar phosphotransferase system (PTS) are present. Inactivation of either alfB or alfF abolishes the growth of L. casei on Fuc-a-l,3-GlcNAc. This proves that AlfB is involved in Fuc-a-1,3-GlcNAc metabolism and that the transporter encoded by alfEFG participates in the uptake of this disaccharide. A mutation in the PTS general component enzyme I does not eliminate the utilization of Fuc-a-1,3-GlcNAc, suggesting that the transport via the PTS encoded by alfEFG is not coupled to phosphorylation of the disaccharide. Transcriptional analysis with alfR and ccpA mutants shows that the two gene clusters alfBR and alfEFG are regulated by substrate-specific induction mediated by the inactivation of the transcriptional repressor AlfR and by carbon catabolite repression mediated by the catabolite control protein A (CcpA). This work reports for the first time the characterization of the physiological role of an a-L-fucosidase in lactic acid bacteria and the utilization of Fuc-a-1,3-GlcNAc as a carbon source for bacteria. [ABSTRACT FROM AUTHOR]

Abstract: UDP-sugars are widely used as substrates in the synthesis of oligosaccharides catalyzed by glycosyltransferases. In the present work a metabolic engineering strategy aimed to direct the carbon flux towards UDP-glucose and UDP-galactose biosynthesis was successfully applied in Lactobacillus casei. The galU gene coding for UDP-glucose pyrophosphorylase (GalU) enzyme in L. casei BL23 was cloned under control of the inducible nisA promoter and it was shown to be functional by homologous overexpression. Notably, about an 80-fold increase in GalU activity resulted in approximately a 9-fold increase of UDP-glucose and a 4-fold increase of UDP-galactose. This suggested that the endogenous UDP-galactose 4-epimerase (GalE) activity, which inter-converts both UDP-sugars, is not sufficient to maintain the UDP-glucose/UDP-galactose ratio. The L. casei galE gene coding for GalE was cloned downstream of galU and the resulting plasmid was transformed in L. casei. The new recombinant strain showed about a 4-fold increase of GalE activity, however this increment did not affect that ratio, suggesting that GalE has higher affinity for UDP-galactose than for UDP-glucose. The L. casei strains constructed here that accumulate high intracellular levels of UDP-sugars would be adequate hosts for the production of oligosaccharides. [Copyright &y& Elsevier]

Abstract: 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 (p <0.05) found between ages. Pigs younger than 10 weeks old and older than 12 weeks old showed high seroprevalences (70–100%), while pigs aged 10–12 weeks showed no detectable serum antibodies levels. Our results suggest that PES infections are common in pigs and that passively acquired maternal antibodies are soon replaced by actively acquired antibodies, whose titers increase gradually with age and that probably are maintained during lifetime. [ABSTRACT FROM AUTHOR]

Abstract: A novel PCR method was developed to discriminate amongst genotypes A–C of the rotavirus non-structural protein 4 (NSP4). Genotype-specific primers were designed that correctly identified the NSP4 genotype when evaluated as a multiplex PCR with cell culture adapted rotavirus strains. Rotavirus strains B223 SGIG6P6[1], NCDV SGIG6P6[1] and SA11 SGIG3P5B[2] were used as control for NSP4 genotype A; A34 SGIG5P14[23], Gottfried SGIIG4P2B[6] and Wa SGIIG1P1A[8] for NSP4 genotype B; RRV SGIG3P5B[3] for NSP4 genotype C. Subsequently, the same set of specific primers was used to genotype a set of 77 Swedish clinical samples. The results showed that all human clinical samples analyzed belong to the NSP4 genotype B and the VP6 subgroup II. [Copyright &y& Elsevier]

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 enzyme immunoassay and in Western blot. Four truncated forms of the NSP4 protein were constructed which allowed us to map the binding region of the selected scFv antibodies to the C-terminal portion of NSP4. The isolated scFv antibodies constitute valuable tools to analyse the mechanisms of NSP4 functions. [Copyright &y& Elsevier]

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 glycoproteins, expressed as polyhistidine-tagged molecules, were analyzed by Western blotting and immunoprecipitation. Newborn mice responded with diarrhea after inoculation with each of the recombinant NSP4 proteins. [Copyright &y& Elsevier]

Rotavirus remains a significant public health threat, especially in low-income countries, where it is the leading cause of severe acute childhood gastroenteritis, contributing to over 128,500 deaths annually. Although the introduction of the Rotarix and RotaTeq vaccines in 2006 marked a milestone in reducing mortality rates, approximately 83,158 preventable deaths persisted, showing ongoing challenges in vaccine accessibility and effectiveness. To address these issues, a novel subcutaneous vaccine formulation targeting multiple rotavirus genotypes has been developed. This vaccine consists of nine VP8* proteins from nine distinct rotavirus genotypes and sub-genotypes (P[4], P[6], P[8]LI, P[8]LIII, P[8]LIV, P[9], P[11], P[14], and P[25]) expressed in E. coli. Two groups of mice were immunized either with a single immunogen, the VP8* from the rotavirus Wa strain (P[8]LI), or with the nonavalent formulation. Preliminary results from mouse immunization studies showed promising outcomes, eliciting antibody responses against six of the nine immunogens. Notably, significantly higher antibody titers against VP8* P[8]LI were observed in the group immunized with the nonavalent vaccine compared to mice specifically immunized against this genotype alone. Overall, the development of parenteral vaccines targeting multiple rotavirus genotypes represents a promising strategy in mitigating the global burden of rotavirus-related morbidity and mortality, offering new avenues for disease prevention and control. [ABSTRACT FROM AUTHOR]

Much evidence suggests a role for human milk oligosaccharides (HMOs) in establishing the infant microbiota in the large intestine, but the response of particular bacteria to individual HMOs is not well known. Here twelve bacterial strains belonging to the genera Bifidobacterium, Enterococcus, Limosilactobacillus, Lactobacillus, Lacticaseibacillus, Staphylococcus and Streptococcus were isolated from infant faeces and their growth was analyzed in the presence of the major HMOs, 2′-fucosyllactose (2′FL), 3-fucosyllactose (3FL), 2′,3-difucosyllactose (DFL), lacto-N-tetraose (LNT) and lacto-N-neo-tetraose (LNnT), present in human milk. Only the isolated Bifidobacterium strains demonstrated the capability to utilize these HMOs as carbon sources. Bifidobacterium infantis Y538 efficiently consumed all tested HMOs. Contrarily, Bifidobacterium dentium strains Y510 and Y521 just metabolized LNT and LNnT. Both tetra-saccharides are hydrolyzed into galactose and lacto-N-triose (LNTII) by B. dentium. Interestingly, this species consumed only the galactose moiety during growth on LNT or LNnT, and excreted the LNTII moiety. Two β-galactosidases were characterized from B. dentium Y510, Bdg42A showed the highest activity towards LNT, hydrolyzing it into galactose and LNTII, and Bdg2A towards lactose, degrading efficiently also 6′-galactopyranosyl-N-acetylglucosamine, N-acetyl-lactosamine and LNnT. The work presented here supports the hypothesis that HMOs are mainly metabolized by Bifidobacterium species in the infant gut. [ABSTRACT FROM AUTHOR]

Fucosyl-oligosaccharides (FUS) provide many health benefits to breastfed infants, but they are almost completely absent from bovine milk, which is the basis of infant formula. Therefore, there is a growing interest in the development of enzymatic transfucosylation strategies for the production of FUS. In this work, the α-L-fucosidases Fuc2358 and Fuc5372, previously isolated from the intestinal bacterial metagenome of breastfed infants, were used to synthesize fucosyllactose (FL) by transfucosylation reactions using p-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as donor and lactose as acceptor. Fuc2358 efficiently synthesized the major fucosylated human milk oligosaccharide (HMO) 2′-fucosyllactose (2′FL) with a 35% yield. Fuc2358 also produced the non-HMO FL isomer 3′-fucosyllactose (3′FL) and traces of non-reducing 1-fucosyllactose (1FL). Fuc5372 showed a lower transfucosylation activity compared to Fuc2358, producing several FL isomers, including 2′FL, 3′FL, and 1FL, with a higher proportion of 3′FL. Site-directed mutagenesis using rational design was performed to increase FUS yields in both α-L-fucosidases, based on structural models and sequence identity analysis. Mutants Fuc2358-F184H, Fuc2358-K286R, and Fuc5372-R230K showed a significantly higher ratio between 2′FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes after 4 h of transfucosylation. The results with the Fuc2358-F184W and Fuc5372-W151F mutants showed that the residues F184 of Fuc2358 and W151 of Fuc5372 could have an effect on transfucosylation regioselectivity. Interestingly, phenylalanine increases the selectivity for α-1,2 linkages and tryptophan for α-1,3 linkages. These results give insight into the functionality of the active site amino acids in the transfucosylation activity of the GH29 α-L-fucosidases Fuc2358 and Fuc5372. Key points: Two α-L-fucosidases from infant gut bacterial microbiomes can fucosylate glycans Transfucosylation efficacy improved by tailored point-mutations in the active site F184 of Fuc2358 and W151 of Fuc5372 seem to steer transglycosylation regioselectivity [ABSTRACT FROM AUTHOR]

Rotavirus (RV) is the leading cause of acute gastroenteritis (AGE) in children under 5 years old worldwide, and several studies have demonstrated that histo–blood group antigens (HBGAs) play a role in its infection process. In the present study, human stool filtrates from patients diagnosed with RV diarrhea (genotyped as P[8]) were used to infect differentiated Caco-2 cells (dCaco-2) to determine whether such viral strains of clinical origin had the ability to replicate in cell cultures displaying HBGAs. The cell culture-adapted human RV Wa model strain (P[8] genotype) was used as a control. A time-course analysis of infection was conducted in dCaco-2 at 1, 24, 48, 72, and 96 h. The replication of two selected clinical isolates and Wa was further assayed in MA104, undifferentiated Caco-2 (uCaco-2), HT29, and HT29-M6 cells, as well as in monolayers of differentiated human intestinal enteroids (HIEs). The results showed that the culture-adapted Wa strain replicated more efficiently in MA104 cells than other utilized cell types. In contrast, clinical virus isolates replicated more efficiently in dCaco-2 cells and HIEs. Furthermore, through surface plasmon resonance analysis of the interaction between the RV spike protein (VP8*) and its glycan receptor (the H antigen), the V7 RV clinical isolate showed 45 times better affinity compared to VP8* from the Wa strain. These findings support the hypothesis that the differences in virus tropism between clinical virus isolates and RV Wa could be a consequence of the different HBGA contents on the surface of the cell lines employed. dCaco-2, HT29, and HT29M6 cells and HIEs display HBGAs on their surfaces, whereas MA104 and uCaco-2 cells do not. These results indicate the relevance of using non-cell culture-adapted human RV to investigate the replication of rotavirus in relevant infection models. [ABSTRACT FROM AUTHOR]

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. Higher levels of IgA against NoV and RV were related to secretor-positive status. No significant differences were found between the FUT2 genotype groups, although the multivariate analysis showed a significant impact of host genotype on specific viral susceptibilities in the microbiome composition. A specific link was found between the abundance of certain bacterial groups, such as Faecalibacterium and Ruminococcus spp., and lower IgA titers against NoV and RV. As a conclusion, we can state that there is a link between host genetics, gut microbiota, and susceptibility to viral infections in humans. [ABSTRACT FROM AUTHOR]

Abstract 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 samples prepared in orange juice, apple juice, horchata, and milk, respectively, were individually mixed with each virus and incubated overnight at 37 °C. Aged-GTE at 5 mg/ml in apple juice reduced MNV infectivity to undetectable levels and from 1.0 to 1.8 log in milk, horchata and orange juice. Aged-GTE at 5 mg/ml in orange juice, apple juice, horchata and milk reduced HAV infectivity by 1.2, 2.1, 1.5, and 1.7 log, respectively. Additionally, aged-GTE at 5 mg/ml in simulated intestinal fluid reduced MNV titers to undetectable levels and reduced HAV infectivity by ca. 2.0 log. The results show a potential for aged-GTE as a suitable natural option for preventive strategies for foodborne viral diseases. Highlights • HuNoVs were very sensitive to aged-GTE treatment at 37 °C. • ISC-RT-qPCR is a useful method to estimate the infectivity of HuNoV after treatment with GTE. • Aged-GTE at 5 mg/ml in different food models reduced MNV and HAV infectivity. • Aged-GTE in SIF reduced MNV titers to undetectable levels and reduced HAV by ca. 2 log. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 the identification of potential bacterial biomarkers, although mechanistic data on the virus-host-microbiota relation are still needed. The identification of the bacterial and HBGA interactions that are exploited by RV and NoV would place the intestinal microbiota as a new target for alternative therapies aimed at preventing and treating viral gastroenteritis. [ABSTRACT FROM AUTHOR]

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, GII.4-1999, GII.4-2004 and GII.4-2006b, to bind to porcine gastric mucin (PGM), human saliva and differentiated human intestinal Caco-2 cells (D-Caco-2 cells). Results: Distinct patterns of saliva binding with the NoV GII.4 variant VLPs were observed, although they bound to D-Caco-2 cells independently of the expression of HBGAs. Monoclonal antibodies against Lewis antigens were able to block the binding of NoV VLPs to saliva, but not to D-Caco-2 cells. Blocking HBGAs on the surface of D-Caco-2 cells with specific monoclonal antibodies did not affect NoV VLP binding to cellular membranes. Co-localisation of Lewis y (Ley) and H-type 2 antigens with NoV VLPs was not observed by immunofluorescence assays. Conclusion: Although the binding of NoV VLPs of GII.4 genotype variants to human saliva samples occur with distinct HBGA binding patterns and can be blocked by antibodies against Lewis antigens, their attachment to D-Caco-2 cells can be mediated by other receptors, which still need further investigation. [ABSTRACT FROM AUTHOR]

Alport syndrome (AS) is a hereditary disorder caused by pathogenic variants in COL4A3, COL4A4, or COL4A5 genes expressing α3, α4, and α5 chains of basement membrane type IV collagen (COL4). The triple-helical α3α4α5(IV) protomer is a major component of the mature glomerular basement membrane (GBM) whose defective formation in AS leads to structural GBM disruption and kidney dysfunction, often resulting in kidney replacement therapy. A genetically intact renal graft exposes the immune system to a non-tolerized α3α4α5(IV) component and an alloimmune response eventually ensues. So far, only IgG alloantibodies reacting against COL4 have been reported in AS alloimmune responses. Here, we report alloimmune glomerulonephritis mediated by IgA antibodies against the non-collagenous C-terminal domain 1 of the α5(IV) chain in a patient with autosomal recessive AS following a second kidney transplantation. The patient presented a not previously described biallelic variant in the COL4A4 gene. Immunological, diagnostic, and clinical implications are discussed. • Alport Syndrome patients may develop post-transplant alloimmune glomerulonephritis. • We report IgA anti-GBM alloantibodies in Alport Syndrome post-kidney transplant. • Anti-GBM disease mediated by IgA antibodies poses a diagnostic challenge. • p.Gly448Val is a new pathogenic variant for Alport Syndrome at COL4A4 gene. [ABSTRACT FROM AUTHOR]

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 auto-transplantation with mouse feces that obtained prior antibiotic treatment. The inoculation of the different mouse groups with a HuNoVs strain (GII.4 Sydney [P16] genotype) showed that the virus replicated more efficiently in animals only treated with antibiotics but not subject to microbiota transplantation. Viral replication in animals receiving fecal microbiota from newborn infants was intermediate, whereas virus excretion in feces from auto-transplanted mice was as low as in the control mice. The analysis of the fecal microbiota by 16S rDNA NGS showed deep variations in the composition in the different mice groups. Furthermore, differences were observed in the gene expression of relevant immunological mediators, such as IL4, CXCL15, IL13, TNFα and TLR2, at the small intestine. Our results suggest that microbiota depletion eliminates bacteria that restrict HuNoVs infectivity and that the mechanism(s) could involve immune mediators. [ABSTRACT FROM AUTHOR]

Fucosyl- N-acetylglucosamine disaccharides are important core structures that form part of human mucosal and milk glyco-complexes. We have previously shown that AlfB and AlfC α-L-fucosidases from Lactobacillus casei are able to synthesize fucosyl-α-1,3-- N-acetylglucosamine (Fuc-α1,3-GlcNAc) and fucosyl-α-1,6- N-acetylglucosamine (Fuc-α1,6-GlcNAc), respectively, in transglycosylation reactions. Here, these reactions were performed in a semipreparative scale, and the produced disaccharides were purified. The maximum yields obtained of Fuc-α1,3-GlcNAc and Fuc-α1,6-GlcNAc were 4.2 and 9.3 g/l, respectively. The purified fucosyl-disaccharides were then analyzed for their prebiotic effect in vitro using strains from the Lactobacillus casei/ paracasei/ rhamnosus group and from Bifidobacterium species. The results revealed that 6 out of 11 L. casei strains and 2 out of 6 L. rhamnosus strains tested were able to ferment Fuc-α1,3-GlcNAc, and L. casei BL87 and L. rhamnosus BL327 strains were also able to ferment Fuc-α1,6-GlcNAc. DNA hybridization experiments suggested that the metabolism of Fuc-α1,3-GlcNAc in those strains relies in an α-L-fucosidase homologous to AlfB. Bifidobacterium breve and Bibidobacterium pseudocatenolatum species also metabolized Fuc-α1,3-GlcNAc. Notably, L-fucose was excreted from all the Lactobacillus and Bifidobacterium strains fermenting fucosyl-disaccharides, except from strains L. rhamnosus BL358 and BL377, indicating that in these latest strains, L-fucose was catabolized. The fucosyl-disaccharides were also tested for their inhibitory potential of pathogen adhesion to human colon adenocarcinoma epithelial (HT29) cell line. Enteropathogenic Escherichia coli (EPEC) strains isolated from infantile gastroenteritis were used, and the results showed that both fucosyl-disaccharides inhibited adhesion to different extents of certain EPEC strains to HT29 cells in tissue culture. [ABSTRACT FROM AUTHOR]

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. Free RNA was the main biological form of SARS-CoV-2 found in plasma. The detection rate for both viral components was associated with viral load at the upper respiratory tract. Median time to SARS-CoV-2-RBD antibody detection was 14 days (range, 4–38) from onset of symptoms. Decreasing antibody levels were observed in parallel to increasing levels of both RNAemia and N-antigenemia, yet overall a fairly modest inverse correlation (Rho = −0.35; P < 0.001) was seen between virus RNAemia and SARS-CoV-2-RBD antibody levels. The data cast doubts on a major involvement of antibodies in virus clearance from the bloodstream within the timeframe examined. [ABSTRACT FROM AUTHOR]

Background: Genome assembly of viruses with high mutation rates, such as Norovirus and other RNA viruses, or from metagenome samples, poses a challenge for the scientific community due to the coexistence of several viral quasispecies and strains. Furthermore, there is no standard method for obtaining whole-genome sequences in non-related patients. After polyA RNA isolation and sequencing in eight patients with acute gastroenteritis, we evaluated two de Bruijn graph assemblers (SPAdes and MEGAHIT), combined with four different and common pre-assembly strategies, and compared those yielding whole genome Norovirus contigs. Results: Reference-genome guided strategies with both host and target virus did not present any advantages compared to the assembly of non-filtered data in the case of SPAdes, and in the case of MEGAHIT, only host genome filtering presented improvements. MEGAHIT performed better than SPAdes in most samples, reaching complete genome sequences in most of them for all the strategies employed. Read binning with CD-HIT improved assembly when paired with different analysis strategies, and more notably in the case of SPAdes. Conclusions: Not all metagenome assemblies are equal and the choice in the workflow depends on the species studied and the prior steps to analysis. We may need different approaches even for samples treated equally due to the presence of high intra host variability. We tested and compared different workflows for the accurate assembly of Norovirus genomes and established their assembly capacities for this purpose. [ABSTRACT FROM AUTHOR]

• Enhanced HIE protocols enabled the detection of infectious gastroenteritis viruses. • Gastroenteritis viruses comparably replicated in single and multiple co-infections. • Replication of HuNoVs, RV, HAstV, and/or HAdV was observed in 57 % of wastewater. • Three wastewater samples exhibited replication of HuNoV GI and GII in HIE. • HIE model confirmed infectivity of various gastroenteritis viruses in wastewater. Fecal-orally transmitted gastroenteritis viruses, particularly human noroviruses (HuNoVs), are a public health concern. Viral transmission risk through contaminated water results underexplored as they have remained largely unculturable until recently and the robust measuring of gastroenteritis viruses infectivity in a single cell line is challenging. This study primarily aimed to test the feasibility of the human intestinal enteroids (HIE) model to demonstrate the infectivity of multiple gastroenteritis viruses in wastewater. Initially, key factors affecting viral replication in HIE model were assessed, and results demonstrated that the reagent-assisted disruption of 3D HIE represents an efficient alternative to syringe pass-through, and the filtering of HuNoV stool suspensions could be avoided. Moreover, comparable replication yields of clinical strains of HuNoV genogroup I (GI), HuNoV GII, rotavirus (RV), astrovirus (HAstV), and adenoviruses (HAdV) were obtained in single and multiple co-infections. Then, the optimized HIE model was used to demonstrate the infectivity of multiple naturally occurring gastroenteritis viruses from wastewater. Thus, a total of 28 wastewater samples were subjected to (RT)-qPCR for each virus, with subsequent testing on HIE. Among these, 16 samples (57 %) showed replication of HuNoVs (n = 3), RV (n = 5), HAstV (n = 8), and/or HAdV (n = 5). Three samples showed HuNoV replication, and sequences assigned to HuNoV GI.3[P13] and HuNoV GII.4[P16] genotypes. Concurrent replication of multiple gastroenteritis viruses occurred in 4 wastewater samples. By comparing wastewater concentrate and HIE supernatant sequences, diverse HAstV and HAdV genotypes were identified in 4 samples. In summary, we successfully employed HIE to demonstrate the presence of multiple infectious human gastroenteritis viruses, including HuNoV, in naturally contaminated wastewater samples. [ABSTRACT FROM AUTHOR]

Whether antibody levels measured by commercially available enzyme or chemiluminescent immunoassays targeting the SARS-CoV-2 spike (S) protein can act as a proxy for serum neutralizing activity remains to be established for many of these assays. We evaluated the degree of correlation between neutralizing antibodies (NtAb) binding the SARS-CoV-2 spike (S) protein and SARS-CoV-2-S-IgG levels measured by four commercial immunoassays in sera drawn from hospitalized COVID-19 patients. Ninety sera from 51 hospitalized COVID-19 patients were tested by a pseudotyped virus neutralization assay, the LIAISON SARS-CoV-2 S1/S2 IgG, the Euroimmun SARS-CoV-2 IgG ELISA, the MAGLUMI 2019-nCoV IgG, and the COVID-19 ELISA IgG assays. Overall, the results obtained with the COVID-19 ELISA IgG test showed the highest agreement with the NtAb assay (κ, 0.85; 95% CI, 0.63–1). The most sensitive tests were the pseudotyped virus NtAb assay and the COVID-19 ELISA IgG assay (92.2% for both). Overall, the degree correlation between antibody titers resulting in 50% virus neutralization (NtAb50) in the pseudotyped virus assay and SARS-CoV-2 IgG levels was strong for the Euroimmun SARS-CoV-2 IgG ELISA (rho = 0.73) and moderate for the remaining assays (rho = 0.48 to 0.59). The kinetic profile of serum NtAb50 titers could not be reliably predicted by any of the SARS-CoV-2 IgG immunoassays. The suitability of SARS-CoV-2-S-IgG commercial immunoassays for inferring neutralizing activity of sera from hospitalized COVID-19 patients varies widely across tests and is influenced by the time of sera collection after the onset of symptoms. [ABSTRACT FROM AUTHOR]

The circulation of Aichi virus in a major urban area was demonstrated using molecular detection with samples recovered from a major river polluted with sewage discharges in Caracas, Venezuela. Five out of 11 water samples studied were positive, being classified by phylogenetic analysis as genotype B. Analysis of sewage waters appears to be a useful methodology to uncover the presence of a hitherto undetected fecal pathogen in a given geographical area. [ABSTRACT FROM AUTHOR]

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 were increased by 3FN fermentation and B. breve by GNB and 3FN. There was a significant reduction of Blautia coccoides group with LNB and 3FN. In addition, 6FN significantly reduced the levels of Enterobacteriaceae family members. Significantly higher concentrations of lactate, formate and acetate were produced in cultures containing either LNB or GNB in comparison with control cultures. Additionally, after fermentation of the oligosaccharides by the fecal microbiota, several Bifidobacterium strains were isolated and identified. The results presented here indicated that each, LNB, GNB and 3FN disaccharide, might have a specific beneficial effect in the infant gut microbiota and they are potential prebiotics for application in infant foods. [ABSTRACT FROM AUTHOR]

The aims of the present work were to determine the prevalence and titer of serum antibodies against several rotavirus VP8* proteins from different P genotypes in children and adults in Valencia, Spain; and to determine the role of the secretor status (FUT2G428A polymorphism) in the antibody response. The VP8* protein from the P[4], P[6], P[8], P[9], P[11], P[14] and P[25] genotypes were produced in E. coli. These proteins were tested with 88 serum samples from children (n = 41, 3.5 years old in average) and from adults (n = 47, 58 years old in average) by ELISA. A subset of 55 samples were genotyped for the FUT2G428A polymorphism and the antibody titers compared. The same subset of samples was also analysed by ELISA using whole rotavirus Wa particles (G1P[8]) as antigen. Ninety-three per cent of the samples were positive for at least one of the VP8* antigens. Differences in the IgG seroprevalence were found between children and adults for the P[4], P[8] and P[11] genotypes. Similarly, significant differences were found between adults and children in their antibody titers against the P[4], P[8], and P[11] VP8* genotypes, having the children higher antibody titers than adults. Interestingly, positive samples against rare genotypes such as P[11] (only in children), P[14] and P[25] were found. While no statistical differences in the antibody titers between secretors and non-secretors were found for any of the tested P genotypes studied, a higher statistic significant prevalence for the P[25] genotype was found in secretors compared to non-secretors. Significant differences in the antibody titers between secretors and non-secretors were found when the whole viral particles from the Wa rotavirus strain (G1P[8]) were used as the antigen. [ABSTRACT FROM AUTHOR]

Rotavirus is the leading agent causing acute gastroenteritis in young children, with the P[8] genotype accounting for more than 80% of infections in humans. The molecular bases for binding of the VP8* domain from P[8] VP4 spike protein to its cellular receptor, the secretory H type-1 antigen (Fuc-α1,2-Gal-β1,3-GlcNAc; H1), and to its precursor lacto-N-biose (Gal-β1,3-GlcNAc; LNB) have been determined. The resolution of P[8] VP8* crystal structures in complex with H1 antigen and LNB and site-directed mutagenesis experiments revealed that both glycans bind to the P[8] VP8* protein through a binding pocket shared with other members of the P[II] genogroup (i.e.: P[4], P[6] and P[19]). Our results show that the L-fucose moiety from H1 only displays indirect contacts with P[8] VP8*. However, the induced conformational changes in the LNB moiety increase the ligand affinity by two-fold, as measured by surface plasmon resonance (SPR), providing a molecular explanation for the different susceptibility to rotavirus infection between secretor and non-secretor individuals. The unexpected interaction of P[8] VP8* with LNB, a building block of type-1 human milk oligosaccharides, resulted in inhibition of rotavirus infection, highlighting the role and possible application of this disaccharide as an antiviral. While key amino acids in the H1/LNB binding pocket were highly conserved in members of the P[II] genogroup, differences were found in ligand affinities among distinct P[8] genetic lineages. The variation in affinities were explained by subtle structural differences induced by amino acid changes in the vicinity of the binding pocket, providing a fine-tuning mechanism for glycan binding in P[8] rotavirus. [ABSTRACT FROM AUTHOR]

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 min) HuNoV GI and GII. This pretreatment reduced the signal of thermally-inactivated HuNoV GI in cockles and HuNoV GII in mussels by > 3 log. Additionally, this pretreatment reduced the signal of thermally-inactivated HuNoV GI and GII between 1 and 1.5 log in oysters. Thermal inactivation of HuNoV GI and GII in PBS, sewage and bioaccumulated oysters was also evaluated by the PMAxx-Triton pretreatment. Results showed significant differences between reductions observed in the control and PMAxx-treated samples in PBS following treatment at 72 and 95 °C for 15 min. In sewage, the RT-qPCR signal of HuNoV GI was completely removed by the PMAxx pretreatment after heating at 72 and 95 °C, while the RT-qPCR signal for HuNoV GII was completely eliminated only at 95 °C. Finally, the PMAxx-Triton pretreatment was applied to naturally contaminated sewage and oysters, resulting in most of the HuNoV genomes quantified in sewage and oyster samples (12 out of 17) corresponding to undamaged capsids. Although this procedure may still overestimate infectivity, the PMAxx-Triton pretreatment represents a step forward to better interpret the quantification of intact HuNoVs in complex matrices, such as sewage and shellfish, and it could certainly be included in the procedures based on RT-qPCR. [ABSTRACT FROM AUTHOR]

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 predicted epitope is composed of 11 amino acids within the P domain: P245, E247, I389, Q390, R397, R435, G443, Y444, P445, N446, and D448. Only two of them, R397 and D448, differ from the homologous variant (GII.4 Den-Haag_2006b) and from a previous variant (GII.4 VA387_1996) that is not recognized by the antibody. A double mutant derived from the VA387_1996 variant containing both changes, Q396R and N447D, is recognized by the 3C3G3 monoclonal antibody, confirming the participation of the two sites in the epitope recognized by the antibody. Furthermore, a single change, Q396R, is able to modify the histo-blood group antigen (HBGA) recognition pattern. These results provide evidence that the epitope recognized by the 3C3G3 antibody is involved in the virus-host interactions, both at the immunological and at the receptor levels. [ABSTRACT FROM AUTHOR]

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 more directly test for hyper-mutation, we performed transfection assays in which the production of mutations was restricted to a single cell infection cycle. This confirmed the presence of sequences with multiple U-to-C/A-to-G transitions, and suggested that hyper-mutation contributed a large fraction of the total NoV spontaneous mutation rate. The type of changes produced and their sequence context are compatible with ADAR-mediated editing of the viral RNA. [ABSTRACT FROM AUTHOR]

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 the NoV GII.4 genotype, 2006b variant, and saliva from a secretor O blood type donor.Results: The most relevant finding was that an asymptomatic non-secretor individual shed NoVs in his stools. Interestingly, anti-NoV IgA antibody titers in saliva from secretor and non-secretor individuals showed no differences. On the contrary, high titers of NoV-specific IgG antibody were found in both convalescent sera and in sera collected 1 year post-infection, but only from secretor individuals. NoV GII.4-2006b VLP binding to receptors present in the saliva was efficiently blocked only by sera from secretor positive individuals.Conclusions: Despite the small number of individuals involved in this outbreak, this study reinforces the idea that susceptibility to human NoV is both dependent on the HBGA profile of the individuals as well as on the viral genotype and variant. We also show that the immunity to NoV lasts for at least 1 year after infection, demonstrating that symptomatic infections strongly stimulate immune responses. [ABSTRACT FROM AUTHOR]

The impact of the COVID-19 pandemic has reinforced the need for rapid, cost-effective, and reliable point-of-care testing (POCT) devices for massive population screening. The co-circulation of SARS-CoV-2 with several seasonal respiratory viruses highlights the need for multiplexed biosensing approaches. Herein, we present a fast and robust all-in-one POCT device for parallel viral antigen and serological analysis. The biosensing approach consists of a functionalized polycarbonate disc-shaped surface with microfluidic structures, where specific bioreagents are immobilized in microarray format, and a portable optoelectronic analyzer. The biosensor quantifies the concentration of viral antigens and specific immunoglobulins G and M for SARS-CoV-2, influenza A/B, adenovirus, and respiratory syncytial virus, using 30 μL of a sample. The semi-automated analysis of 6 samples is performed in 30 min. Validation studies performed with 135 serum samples and 147 nasopharyngeal specimens reveal high diagnostic sensitivity (98–100%) and specificity (84–98%), achieving an excellent agreement (κ = 0.937) with commercial immunoassays, which complies with the World Health Organization criteria for POC COVID-19 diagnostic tests. The versatility of the POCT device paves the way for the detection of other pathogens and analytes in the incoming post-pandemic world, integrating specific bioreagents against different variants of concerns and interests. [ABSTRACT FROM AUTHOR]

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 (probiotic and non-probiotic) and a Gram-negative probiotic strain have the ability to bind P-particles on their surfaces irrespective of their probiotic status. The binding of P-particles (GI.1) to HT-29 cells in the presence of selected strains showed that bacteria can inhibit P-particle attachment in competitive exclusion experiments. However, pre-treatment of cells with bacteria or adding bacteria to cells with already attached P-particles enhanced the retention of the particles. Although direct viral binding and blocking of viral receptors have been postulated as mechanisms of protection against viral infection by probiotic bacteria, these results highlight the need for a careful evaluation of this hypothesis. The work presented here investigates for the first time the probiotic-NoVs-host interactions and points up the NoVs P-particles as useful tools to overcome the absence of in vitro cellular models to propagate these viruses. [ABSTRACT FROM AUTHOR]

Abstract: Heparanase is a key enzyme involved in the dissemination of metastatic cancer cells. In this study a combination of in silico techniques and experimental methods was used to identify new potential inhibitors against this target. A 3D model of heparanase was built from sequence homology and applied to the virtual screening of a library composed of 27 known heparanase inhibitors and a commercial collection of drugs and drug-like compounds. The docking results from this campaign were combined with those obtained from a pharmacophore model recently published based in the same set of chemicals. Compounds were then ranked according to their theoretical binding affinity, and the top-rated commercial drugs were selected for further experimental evaluation. Biophysical methods (NMR and SPR) were applied to assess experimentally the interaction of the selected compounds with heparanase. The binding site was evaluated via competition experiments, using a known inhibitor of heparanase. Three of the selected drugs were found to bind to the active site of the protein and their K D values were determined. Among them, the antimalarial drug amodiaquine presented affinity towards the protein in the low-micromolar range, and was singled out for a SAR study based on its chemical scaffold. A subset of fourteen 4-arylaminoquinolines from a global set of 249 analogues of amodiaquine was selected based on the application of in silico models, a QSAR solubility prediction model and a chemical diversity analysis. Some of these compounds displayed binding affinities in the micromolar range. [Copyright &y& Elsevier]

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 bound the authentic outbreak GII.4 Valencia/2004/Es virus, but did not in contrast to secretor-positive saliva bind VLP of other strains including the GII.4 Dijon strain. Amino acid comparison of antigenic A and B sites located on the external loops of the P2 domain revealed distinct differences between the Valencia/2004/Es and Dijon strains. All three aa in each antigenic site as well as 10/11 recently identified evolutionary hot spots, were unique in the Valencia/2004/Es strain compared to the Dijon strain. To the best of our knowledge, this is the first example of symptomatic GII.4 norovirus infection of a Lea+b- individual homozygous for the G428A nonsense mutation in FUT2. Taken together, our study provides new insights into the host genetic susceptibility to norovirus infections and evolution of the globally dominating GII.4 viruses. [ABSTRACT FROM AUTHOR]

Rotavirus (RV) and norovirus (NoV) are the leading causes of acute gastroenteritis (AGE) worldwide. Several studies have demonstrated that histo-blood group antigens (HBGAs) have a role in NoV and RV infections since their presence on the gut epithelial surfaces is essential for the susceptibility to many NoV and RV genotypes. Polymorphisms in genes that code for enzymes required for HBGAs synthesis lead to secretor or non-secretor and Lewis positive or Lewis negative individuals. While secretor individuals appear to be more susceptible to RV infections, regarding NoVs infections, there are too many discrepancies that prevent the ability to draw conclusions. A second factor that influences enteric viral infections is the gut microbiota of the host. In vitro and animal studies have determined that the gut microbiota limits, but in some cases enhances enteric viral infection. The ways that microbiota can enhance NoV or RV infection include virion stabilization and promotion of virus attachment to host cells, whereas experiments with microbiota-depleted and germ-free animals point to immunoregulation as the mechanism by which the microbiota restrict infection. Human trials with live, attenuated RV vaccines and analysis of the microbiota in responder and non-responder individuals also allowed the identification of bacterial taxa linked to vaccine efficacy. As more information is gained on the complex relationships that are established between the host (glycobiology and immune system), the gut microbiota and intestinal viruses, new avenues will open for the development of novel anti-NoV and anti-RV therapies. [ABSTRACT FROM AUTHOR]

Background. Histo-blood group antigens and secretor status have been associated with susceptibility to Norovirus infections, which suggests that antibody prevalence and titer might correlate with these phenotypes. Methods. Plasma samples (n = 105) from Swedish blood donors that had been genotyped for secretor (FUT2) and Lewis (Le; FUT3) genotypes and phenotyped for ABO and Le blood groups were analyzed for immunoglobulin G antibody prevalence and titers to norovirus genogroup (GG) II.4. Results. The results showed that nonsecretors (se428se428) and Lea+b- individuals not only had significantly lower antibody titers than did secretors (P < .0001) and Lea-b+ individuals (P < .0002) but were also significantly more often antibody negative (P < .05). Antibody titers in secretors were not significantly different between individuals of different Le (FUT3) genotypes or different ABO phenotypes. Conclusions. Nonsecretors and Lea+b- individuals are significantly less prone to be infected with GGII noroviruses. This new information extends previous knowledge and supports the hypothesis that nonsecretors are relatively but not absolutely resistant to norovirus infections. [ABSTRACT FROM AUTHOR]

• SARS-CoV-2-S1/M-reactive-IFN-γ CD4+ and CD8+ T cells were detected in less than 30% recovered patients from severe COVID-19. • 60% of individuals displayed detectable SARS-CoV-2-RBD-specific IgGs at 2–5 months following COVID-19 diagnosis. • The presence of comorbidities may hamper persistence of SARS-CoV-2 -S1/M-reactive T cells in recovered COVID-19 patients. There 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. We 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 heparinized whole blood by flow cytometry for ICS. Detectable SARS-CoV-2-S1/M-reactive CD69+-IFN-γ CD4+ and CD8+ T cells were displayed in 17 (29.3%) and 6 (10.3%) subjects respectively, at a median of 84 days after onset of symptoms (range, 58–191 days). Concurrent comorbidities increased the risk (OR, 3.15; 95% CI, 1.03–9.61; P = 0.04) of undetectable T–cell responses in models adjusted for age, sex and hospitalization ward. Twenty-one out of the 35 patients (60%) had detectable RBD-specific serum IgGs at a median of 118 days (range, 60–145 days) after symptoms onset. SARS-CoV-2 RBD-specific IgG serum levels were found to drop significantly over time. A relatively limited number of subjects who developed severe forms of COVID-19 had detectable SARS-CoV-2-S1/M IFNγ CD4+ and CD8+ T cells at midterm after clinical diagnosis. Our data also indicated that serum levels of RBD-specific IgGs decline over time, becoming undetectable in some patients. [ABSTRACT FROM AUTHOR]

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 (12.2%) and ahead of rotavirus (7.1%), astrovirus (4.9%) and enteric adenoviruses (2.9%). Most sapovirus infections occurred in infants and young children under 3 years of age (74%) with the highest prevalence in autumn and early winter. Coinfections were found in 25% of the patients with sapovirus diarrhea, mainly with other enteric viruses. Genotyping demonstrated the circulation of seven different genotypes during the study period, with a predominance of genotypes GI.1, GI.2, and GII.1. Phylogenetic analysis showed that genogroup GII strains form a cluster separated from genogroup GI and GV, being genotype GV.1 strains related to genotype GI.1 and GI.2 strains. [ABSTRACT FROM AUTHOR]

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 4.8- and 5.4-fold compared to samples nonlabeled with anti-RV antibodies. In vitro binding of the G1P[8] Wa human RV strain to two Ruminococcus gauvreauii human isolates was confirmed by fluorescence microscopy. Analysis in R. gauvreauii with antibodies directed to several histo-blood group antigens (HBGAs) indicated that these bacteria express HBGA-like substances on their surfaces, which can be the target for RV binding. Furthermore, in vitro infection of the Wa strain in differentiated Caco-2 cells was significantly reduced by incubation with R. gauvreauii. These data, together with previous findings showing a negative correlation between Ruminococcus levels and antibody titers to RV in healthy individuals, suggest a pivotal interaction between this bacterial group and human RV. These results reveal likely mechanisms of how specific bacterial taxa of the intestinal microbiota could negatively affect RV infection and open new possibilities for antiviral strategies. [ABSTRACT FROM AUTHOR]

• Neutralizing antibodies targeting SARS-CoV-2 S protein correlate with IgGs binding RBD. • NtAb and RBD IgGs levels do not associate with COVID-19 severity in hospitalized patients. • NtAb and RBD IgG levels do not correlate with serum levels of inflammatory biomarkers. 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. 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 were measured in a GFP reporterbased pseudotyped virus platform. Demographic and clinical data, complete blood counts, as well as serum levels of ferritin, Dimer-D, C reactive protein (CRP), lactose dehydrogenase (LDH), and interleukin-6 (IL-6) were retrieved from clinical charts. The overall correlation between levels of both antibody measurements was good (Rho = 0.82; P = 0 < 0.001). SARS-CoV-2 RBD IgG and NtAb50 levels in sera collected up to day 30 after the onset of symptoms were comparable between ICU and non-ICU patients (P=>0.1). Four ICU patients died; two of these achieved NtAb50 titers ≥1/160 while the other two exhibited a 1/80 titer. Very weak (Rho=>0.0–<0.2) or weak (Rho=>0.2–<0.4) correlations were observed between anti-RBD IgGs, NtAb50, and serum levels pro-inflammatory biomarkers. The data presented herein do not support an association between SARS-CoV-2 RBD IgG or NtAb50 levels and COVID-19 severity. [ABSTRACT FROM AUTHOR]

Background: Human group A rotavirus is the leading cause of severe acute gastroenteritis in young children worldwide. Immunization programs have reduced the disease burden in many countries. Vaccination coverage in the Autonomous Region of Valencia, Spain, is around 40%, as the rotavirus vaccine is not funded by the National Health System. Despite this low-medium vaccine coverage, rotavirus vaccination has substantially reduced hospitalizations due to rotavirus infection and hospital-related costs. However, there are very few studies evaluating symptomatic rotavirus infections not requiring hospitalization in vaccinated children. The objective of this study was to investigate symptomatic rotavirus infections among vaccinated children in the health area served by the Hospital Clínico Universitario of Valencia, Spain, from 2013 to 2015.Methods: A total of 133 children younger than 5 years of age with rotavirus infection were studied. Demographic and epidemiological data were collected and informed consent from their caretakers obtained. Rotavirus infection was detected by immunological methods and G/P rotavirus genotypes were determined by RT-PCR, following standard procedures from the EuroRotaNet network.Results: Forty infants (30.1%; 95% CI: 22.3-37.9) out of 133 were diagnosed with symptomatic rotavirus infection despite having been previously vaccinated, either with RotaTeq (85%) or with Rotarix (15%). Children fully vaccinated against rotavirus (24.8%), partially vaccinated (5.3%) and unvaccinated (69.9%) were found. The infecting genotypes showed high G-type diversity, although no significant differences were found between the G/P genotypes infecting vaccinated and unvaccinated children during the same time period. G9P[8], G12P[8] and G1P[8] were the most prevalent genotypes. Severity of gastroenteritis symptoms required 28 (66.6%) vaccinated and 67 (73.6%) unvaccinated children to be attended at the Emergency Room.Conclusion: Rotavirus vaccine efficacy in reducing the incidence of severe rotavirus infection has been well documented, but symptomatic rotavirus infection can sometimes occur in vaccinees. [ABSTRACT FROM AUTHOR]

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 (2013–2015), stool and saliva samples were collected from 133 children with rotavirus infection. Infecting viral genotypes and HBGAs were determined in patients and compared to a control group and data from blood donors. Rotavirus G9P[8] was the most prevalent strain (49.6%), followed by G1P[8] (20.3%) and G12P[8] (14.3%). Rotavirus infected predominantly secretor (99%) and Lewis b positive (91.7%) children. Children with blood group A and AB were significantly more prone to rotavirus gastroenteritis than those with blood group O. Our results confirm that a HBGA genetic background is linked to rotavirus P[8] susceptibility. Rotavirus P[8] symptomatic infection is manifestly more frequent in secretor-positive (FUT2+) than in non-secretor individuals, although no differences between rotavirus G genotypes were found. [ABSTRACT FROM AUTHOR]