Aproximaciones moleculares al estudio de las interacciones de rotavirus y norovirus con el huésped

Los norovirus humanos son los principales agentes etiológicos de gastroenteritis aguda en todo el mundo. Aunque los norovirus son muy diversos (se han detectado más de 30 genotipos en humanos), durante los últimos 40 años la mayoría de los brotes y epidemias han sido causados por cepas del genotipo GII.4, lo que plantea interrogantes sobre su persistencia en la población. Entre otras posibles explicaciones, se considera que la evasión inmunológica es la principal causa de su éxito. Para estudiar en detalle el reconocimiento y la evasión de anticuerpos, analizamos un epítopo conformacional reconocido por un anticuerpo monoclonal (3C3G3) mediante mutagénesis dirigida y resonancia superficial de plasmón (SPR, por sus siglas en inglés). Los resultados muestran que el epítopo predicho está compuesto por 11 aminoácidos dentro del dominio P de la cápside vírica. Sólo dos de ellos difieren de la variante homóloga (GII.4 Den-Haag_2006b) y de la variante pre- epidémica (GII.4 VA387_1996) que no es reconocida por el anticuerpo. Además, un solo cambio de aminoácido es capaz de modificar el patrón de reconocimiento de los antígenos histo-sanguíneos (HBGA). Estos resultados proporcionan evidencia de que el epítopo reconocido por el anticuerpo 3C3G3 está involucrado en las interacciones virus-huésped, tanto a nivel inmunológico como en el reconocimiento de los receptores celulares. Los rotavirus humanos son los principales agentes causantes de gastroenteritis aguda en niños pequeños, y además el genotipo P[8] representa más del 80% de las infecciones en humanos. Se han determinado las bases moleculares de la unión del dominio VP8* de la proteína VP4 del genotipo P[8] asu receptor celular, el antígeno H tipo 1 (Fuc- " 1,2-Gal- # 1,3-GlcNAc; H1) y a su precursor lacto-N-biosa (Gal- # 1,3-GlcNAc; LNB). La resolución de las estructuras cristalinas de P[8] en complejos con el antígeno H1 y LNB y los experimentos de mutagénesis dirigida revelaron que ambos glicanos se unen a la proteína P[8] VP8* a través de un sitio de unión compartido con otros miembros del genogrupo P[II], es decir; P[4], P[6] y P[19]. Los resultados muestran que la fracción L-fucosa de H1 sólo muestra contactos indirectos con P[8] VP8*. Sin embargo, los cambios conformacionales inducidos en la fracción de la LNB duplican la afinidad del ligando, según las medidas por SPR lo que proporciona una explicación molecular a la diferente susceptibilidad a la infección por rotavirus entre individuos secretores y no secretores. La inesperada interacción de P[8] VP8* con la LNB, un componente básico de los oligosacáridos de la leche humana de tipo 1, dio lugar a la inhibición de la infección por rotavirus, lo que pone de manifiesto el papel y la posible aplicación de este disacárido como antiviral. A pesar de que los aminoácidos clave en el sitio de unión H1/LNB están altamente conservados en los miembros del genogrupo P[II], se han encontrado diferencias en las afinidades de ligandos entre los distintos linajes de P[8]. La variación en las afinidades se explica por sutiles diferencias estructurales inducidas por cambios en los aminoácidos de las proximidades al sitio de unión, que proporcionan un mecanismo de ajuste para la unión del glicano en el bolsillo de unión a la VP8* de los rotavirus del genotipo P[8]. Un último objetivo del presente trabajo fue determinar la prevalencia y el título de los anticuerpos séricos frente a varios genotipos de la proteína VP8* de rotavirus en niños y adultos en Valencia, España, y determinar el papel del estado secretor (polimorfismo FUT2G428A) en la respuesta serológica. Se utilizaron las proteína VP8* de los genotipos P[4], P[6], P[8], P[9], P[11], P[14] y P[25] que fueron analizadas por ELISA con 88 muestras de sueros de niños (n=41, 3.5 años de edad en promedio) y de adultos (n=47, 58 años de edad en promedio). El conjunto de 55 muestras fueron genotipadas para el polimorfismo FUT2G428A y se compararon los títulos de anticuerpos. Se encontraron diferencias en la seroprevalencia entre niños y adultos para los genotipos P[4], P[8] y P[11]. De manera similar, se encontraron diferencias significativas entre adultos y niños en los títulos de anticuerpos contra los genotipos P[4], P[8] y P[11], teniendo los niños títulos de anticuerpos más altos que los adultos. Curiosamente, se encontraron muestras positivas para genotipos raros como P[11] (sólo en niños), P[14] y P[25]. Aunque no se encontraron diferencias estadísticas en los títulos de anticuerpos entre secretores y no secretores para ninguno de los genotipos P estudiados, se encontró una prevalencia estadísticamente significativa más alta para el genotipo P[25] en los individuos secretores en comparación con los no secretores. Finalmente, para profundizar en el estudio de la susceptibilidad entre secretores y no secretores se estudió el titulo de anticuerpos frente al rotavirus completo de la cepa Wa G1P[8], encontrándose diferencias estadísticamente significativas en este caso

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. 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

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 genotyping based on sequencing of the open reading frame 1 (ORF1)/ORF2 junction region, we detected 25 different capsid-polymerase-type associations. The most common GII capsid genotype was GII.4 Sydney 2012, followed by GII.2, GII.3, GII.6, and GII.17. A high prevalence of recombinant strains (90.4%) was observed among GII infections between 2018 and 2020. GII.4 Sydney[P16] was the predominant genotype from 2019 to 2020. In addition, GII.P16 polymerase was found harbored within six different capsid genes. GI.4 and GI.3 were the predominant genotypes in genogroup I, in which recombinant strains were also found, such as GI.3[P10], GI.3[P13], and GI.5[P4]. Interestingly, applying the criterion of 2 times the standard deviation, we found that 12 sequences initially classified as GI.3 may represent two new tentative genotypes in genogroup I, designated GI.10 and GI.11. This study shows the extensive diversity of recombinant noroviruses circulating in Spain and highlights the role of recombination events in the spread of noroviruses

Epidemiological Surveillance of Norovirus and Rotavirus in Sewage (2016-2017) in Valencia (Spain)

The aim of the present study was to perform the molecular epidemiology of rotaviruses and noroviruses detected in sewage samples from a large wastewater facility from the city of Valencia, Spain. A total of 46 sewage samples were collected over a one-year period (September 2016 to September 2017). Norovirus and rotavirus were detected and quantified by RT-qPCR, genotyped by semi-nested RT-PCR and further characterized by sequencing and phylogenetic analyses. Noroviruses and rotaviruses were widely distributed in sewage samples (69.6% for norovirus GI, 76.0% norovirus GII, and 71.7% rotaviruses) and viral loads varied from 4.33 to 5.75 log PCRU/L for norovirus GI, 4.69 to 6.95 log PCRU/L for norovirus GII, and 4.08 to 6.92 log PCRU/L for rotavirus. Overall, 87.5% (28/32) of GI noroviruses could not be genotyped, 6.25% (2/32) of the samples contained GI.2 genotype, and another 6.25% (2/32) were positive for GI.4 genotype. The most common genotype of GII noroviruses was GII.2 (40%, 14/35), followed by GII.6 (8.6%, 3/35) and GII.17 (5.7%, 2/35) while the remaining GII strains could not be typed (45.7%, 16/35). Rotavirus VP4 genotype P[8] was the only one found in 19 out of 33 rotavirus-positive samples (57.7%). G2 was the most prevalent rotavirus VP7 genotype (15.2%, 5/33) followed by G3, G9, and G12, with two positive samples for each genotype (6.1%, 2/33). In one sample both G1 and G2 genotypes were detected simultaneously (3%). The results presented here show that the surveillance of noroviruses and rotaviruses in sewage is useful for the study of their transmission in the population and their molecular epidemiology

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 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

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-establishment of specific bacterial taxons was evidenced. Microbial composition analysis revealed profound changes in the intestinal microbiota of antibiotic-treated animals, whereas some bacterial groups, including members of Lactobacillus, Bilophila, Mucispirillum, and Oscillospira, recovered after self-FMT. In antibiotic-treated and FMT animals where the virus replicated more efficiently, differences were observed in gene expression of immune mediators, such as IL1β and CXCL15, as well as in the fucosyltransferase FUT2, responsible for H-type antigen synthesis in the small intestine. Collectively, our results suggest that antibiotic-induced microbiota depletion eradicates the microbial taxa that restrict human rotavirus infectivity in mice

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

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

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

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