2.2. HBGAs and NoV
The P2 subdomain found in the P-domain of the VP1 protein from NoVs interacts with HBGAs. Several studies have been made in order to elucidate the recognition pattern of NoVs. Some of them are based on the expression of the P-domain in vitro, which results in dimerization (P dimer) and the formation of P particles that retain HBGA-binding function, while others used virus-like particles (VLPs). These studies have utilized ELISA or haemagglutination-based assays using saliva, human milk, red blood cells, or synthetic oligosaccharides as HBGAs sources. The prototype Norwalk virus (GI.1) recognizes the type A and H secretors, but does not interact with type B secretors and non-secretors; Va387 (GII.4) binds to A, B, and O secretors; MOH (GII.5) and Hiro (GII.12) bind to A and B secretors; and Va207 (GII.9) recognizes Lewis positive secretors and non-secretors (Le
x and Le
y)
[18,51][6][24]. As for the GII.4 strains Den Haag_2006b and Sydney_2012, Carmona et al. demonstrated that they did not recognize any HBGAs
[52][25]. By contrast, these strains may recognize heparan sulphate or citrate since they are all capable of binding human NoV and may potentially play a role in NoV pathogenesis as cellular receptors/co-factors
[53][26]. GI.3 NoV VLPs show strong binding to blood type A salivary HBGAs, slightly lower binding to blood type O salivary HBGAs, and weakly binding or none to blood type B and AB salivary HBGAs
[54][27].
3. The Role of Bacteria in RVs and NoVs Infection
3.1. Bacteria against Enteric Viral Infections
Several studies demonstrate the beneficial effect of probiotic bacteria against enteric virus infections and many other diseases
[66,67,68,69][28][29][30][31]. Probiotics protect the host from viral infection by modulating gut microbiota composition, enhancing intestinal barrier function, and promoting mucosal immunity
[70][32]. Additionally, they interfere with the binding of the virus to their target cells by competitive exclusion by blocking viral receptors and binding viruses on the surface to promote their elimination in faeces
[71][33].
3.2. Microbiota and Promotion of Enteric Viral Infections
Despite the significant evidence available about the role of intestinal-derived bacteria in the inhibition of viral infections, several investigations argued for a role of microbiota in promoting virus infection
[81][34]. This was first demonstrated by Kuss et al.
[82][35] and Kane et al.
[83][36] when using poliovirus, reovirus, and mouse mammary tumour virus (MMTV) for infecting germ-free or antibiotic-treated mice. In these cases, it was observed that a substantial attenuation of infection occurred when compared to infection of microbially-colonized mice. Reconstitution of intestinal microorganisms into antibiotic-treated mice was enough to restore poliovirus pathogenesis
[82][35]. Moreover, intestinal titres of reovirus were substantially reduced in antibiotic-treated, compared with control mice
[83][36]. Similar findings were reported with RV and NoV when antibiotic-treated or germ-free mice were used
[84[37][38],
85], suggesting that microbiota enhances the pathogenesis of multiple families of enteric viruses.
3.3. Microbiota and Restriction of Enteric Viral Infections
Recently, it has been shown that microbiota ablation with antibiotics in mice allows for infection with the human RV strain Wa (G1P[8]), which replicates very inefficiently in animals with normal microbiota
[96][39]. These results are in conflict with earlier experiments which demonstrated that microbiota eradication by antibiotics results in reduced infection of murine RV (EC strain), as shown by lower viral shedding in adult mice and diminished diarrhea incidence in mice pups
[84][37]. Nevertheless, viral clearance lasted longer in this model. Furthermore, recent experiments with the murine EDIM strain confirmed that antibiotic treatment and the consequent decrease in intestinal bacterial loads or the use of germ-free mice results in increased RV infection
[97][40], which argues against a positive effect of the microbiota in RV infection. In the experiments with the Wa strain, animals with ablated microbiota and subsequent subjection to self-transplantation of intestinal microbiota partially recovered the resistance to infection, which allowed the identification of bacterial taxa that likely participate indirectly or directly in the restriction of Wa infection in mice
[96][39]. Thus, bacteria belonging to lactobacilli,
Mucispirillum,
Oscillospira, and
Bilophila genera were negatively linked to RV infection in mice. Faecal material transplantation with infants as donors did not restrict infectivity in this model, suggesting that the microbiota from the donors was not able to control RV infection in this model and that mice autochthonous bacteria were needed for the process
[96][39].
4. Microbiota and Enteric Viruses, Studies in Humans
Microbiota composition varies depending on the population [105][41] since it is affected by many factors including nutrition [106][42], sex, age, genetics, and health status [107][43], and these vary greatly between low-income and high-income countries. Such differences could be some of the reasons why RVVs have significantly lower efficacy in low-income countries [108,109][44][45].
A recent study evaluated whether microbiota modification by the use of broad- and narrow-spectrum antibiotics had an effect on immunization with Rotarix in adults [116][46]. Although the experimental groups did not differ in terms of total IgA produced, the narrow spectrum group showed a boost in IgA at day seven post-vaccination (basal levels of anti-RV IgA were high in the vaccination group) and the viral shedding was increased in both groups treated with antibiotics. Differences in the microbiota composition in faeces were evident between the groups and correlations between enrichment in Bacteroides populations at the boost at day seven were observed and several taxa (Prevotellaceae, Cloacibacillus everynsis, and Proteobacteria members such as Escherichia and Shigella) were associated with increased viral shedding. Antibiotic treatment had no effect on the immunogenicity of other systemic vaccines applied (pneumococcal and tetanus vaccine) [116][46]. These results highlight the fact that targeting the microbiota could be an alternative strategy to enhance RVVs efficacy, although the effectiveness in children still needs further investigation.
An ex vivo study analysed the bacterial groups that were interacting with RV in stool samples from children suffering RV (G1P[8]) diarrhea by flow cytometry followed by 16S rDNA sequencing
[80][47]. This study also allowed the identification of
Ruminococcus as RV-interacting bacteria. As already mentioned, a species of this group (
R. gauvreauii) was shown to inhibit RV infection in vitro
[80][47]. This, together with the correlation
Ruminococcus-anti-RV IgA in humans and the fact that higher
Ruminococcus numbers are found in healthy children compared to children with RV diarrhea
[121][48], postulates these bacteria as likely players in the cross-talk bacteria-virus-host. Similar studies conducted with individuals suffering AGE caused by NoV will certainly aid in identifying bacterial taxons that interact with these viruses in stools. However, whether this interaction has some relevance in the infection process needs further investigation.
All these findings may help to improve RVVs performance in such a way that they have higher efficacy in low-income countries, preventing tens of thousands of RV-related deaths per year. However, differences in the conclusions drawn from the microbiota analyses are evident, and standardized and controlled methods (e.g., sampling and DNA extraction techniques, bacterial 16S rDNA sequencing platforms, microbial composition analysis methods, etc.) are needed to get a clearer picture.