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

Rotavirus symptomatic infection among unvaccinated and vaccinated children in Valencia, Spain

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

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 (12.2%) 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