Interferons and tuft cell numbers are bottlenecks for persistent murine norovirus infection.

Noroviruses (NoVs) are a leading cause of viral gastroenteritis. Despite global clinical relevance, our understanding of how host factors, such as antiviral cytokines interferons (IFNs), modulate NoV population dynamics is limited. Murine NoV (MNoV) is a tractable in vivo model for the study of host regulation of NoV. A persistent strain of MNoV, CR6, establishes a reservoir in intestinal tuft cells for chronic viral shedding in stool. However, the influence of host innate immunity and permissive cell numbers on viral population dynamics is an open question. We generated a pool of 20 different barcoded viruses (CR6BC) by inserting 6-nucleotide barcodes at the 3' position of the NS4 gene and used this pool as our viral inoculum for in vivo infections of different mouse lines. We found that over the course of persistent CR6 infection, shed virus was predominantly colon-derived, and viral barcode richness decreased over time irrespective of host immune status, suggesting that persistent infection involves a series of reinfection events. In mice lacking the IFN-λ receptor, intestinal barcode richness was enhanced, correlating with increased viral intestinal replication. IL-4 treatment, which increases tuft cell numbers, also increased barcode richness, indicating the abundance of permissive tuft cells to be a bottleneck during CR6 infection. In mice lacking type I IFN signaling (Ifnar1-/-) or all IFN signaling (Stat1-/-), barcode diversity at extraintestinal sites was dramatically increased, implicating different IFNs as critical bottlenecks at specific tissue sites. Of interest, extraintestinal barcodes were overlapping but distinct from intestinal barcodes, indicating that disseminated virus represents a distinct viral population than that replicating in the intestine. Barcoded viruses are a valuable tool to explore the influence of host factors on viral diversity in the context of establishment and maintenance of infection as well as dissemination and have provided important insights into how NoV infection proceeds in immunocompetent and immunocompromised hosts.

Differentiation and Protective Capacity of Virus-Specific CD8+ T Cells Suggest Murine Norovirus Persistence in an Immune-Privileged Enteric Niche.

Noroviruses can establish chronic infections with active viral shedding in healthy humans but whether persistence is associated with adaptive immune dysfunction is unknown. We used genetically engineered strains of mouse norovirus (MNV) to investigate CD8+ T cell differentiation during chronic infection. We found that chronic infection drove MNV-specific tissue-resident memory (Trm) CD8+ T cells to a differentiation state resembling inflationary effector responses against latent cytomegalovirus with only limited evidence of exhaustion. These MNV-specific Trm cells remained highly functional yet appeared ignorant of ongoing viral replication. Pre-existing MNV-specific Trm cells provided partial protection against chronic infection but largely ceased to detect virus within 72 hours of challenge, demonstrating rapid sequestration of viral replication away from T cells. Our studies revealed a strategy of immune evasion by MNV via the induction of a CD8+ T cell program normally reserved for latent pathogens and persistence in an immune-privileged enteric niche.

Propagating and banking genetically diverse human sapovirus strains using a human duodenal cell line: investigating antigenic differences between strains.

There are four genogroups and 18 genotypes of human sapoviruses (HuSaVs) responsible for acute gastroenteritis. To comprehend their antigenic and virological differences, it is crucial to obtain viral stocks of the different strains. Previously, we utilized the human duodenum-derived cell line HuTu80, and glycocholate, a conjugated bile acid, to replicate and propagate GI.1, GI.2, and GII.3 HuSaVs (H. Takagi et al., Proc Natl Acad Sci U S A 117:32078-32085, 2020, https://10.1073/pnas.2007310117). First, we investigated the impact of HuTu80 passage number on HuSaV propagation. Second, we demonstrated that taurocholate improved the initial replication success rate and viral RNA levels in fecal specimens relative to glycocholate. By propagating 15 HuSaV genotypes (GI.1-7, GII.1-5, -8, and GV.1-2) and accomplishing preparation of viral stocks containing 1.0 × 109 to 3.4 × 1011 viral genomic copies/mL, we found that all strains required bile acids for replication, with GII.4 showing strict requirements for taurocholate. The deduced VP1 sequences of the viruses during the scale-up of serial passaged virus cultures were either identical or differed by only two amino acids from the original sequences in feces. In addition, we purified virions from nine strains of different genotypes and used them as immunogens for antiserum production. Enzyme-linked immunosorbent assays (ELISAs) using rabbit and guinea pig antisera for each of the 15 strains of different genotypes revealed distinct antigenicity among the propagating viruses across genogroups and differences between genotypes. Acquisition of biobanked viral resources and determination of key culture conditions will be valuable to gain insights into the common mechanisms of HuSaV infection. IMPORTANCE: The control of human sapovirus, which causes acute gastroenteritis in individuals of all ages, is challenging because of its association with outbreaks similar to those caused by human norovirus. The establishment of conditions for efficient viral propagation of various viral strains is essential for understanding the infection mechanism and identifying potential control methods. In this study, two critical factors for human sapovirus propagation in a conventional human duodenal cell line were identified, and 15 strains of different genotypes that differed at the genetic and antigenic levels were isolated and used to prepare virus stocks. The preparation of virus stocks has not been successful for noroviruses, which belong to the same family as sapoviruses. Securing virus stocks of multiple human sapovirus strains represents a significant advance toward establishing a reliable experimental system that does not depend on limited virus-positive fecal material.

Human norovirus cultivation systems and their use in antiviral research.

Human norovirus (HuNoV) is a major cause of acute gastroenteritis and foodborne diseases, affecting all age groups. Despite its clinical needs, no approved antiviral therapies are available. Since the discovery of HuNoV in 1972, studies on anti-norovirals, mechanism of HuNoV infection, viral inactivation, etc., have been hampered by the lack of a robust laboratory-based cultivation system for HuNoV. A recent breakthrough in the development of HuNoV cultivation systems has opened opportunities for researchers to investigate HuNoV biology in the context of de novo HuNoV infections. A tissue stem cell-derived human intestinal organoid/enteroid (HIO) culture system is one of those that supports HuNoV replication reproducibly and, to our knowledge, is most widely distributed to laboratories worldwide to study HuNoV and develop therapeutic strategies. This review summarizes recently developed HuNoV cultivation systems, including HIO, and their use in antiviral studies.

Bile acid-sensitive human norovirus strains are susceptible to sphingosine-1-phosphate receptor 2 inhibition.

Human noroviruses (HuNoVs) are a diverse group of RNA viruses that cause endemic and pandemic acute viral gastroenteritis. Previously, we reported that many HuNoV strains require bile or bile acid (BA) to infect human jejunal intestinal enteroid cultures. BA was not essential for the replication of a pandemic-causing GII.4 HuNoV strain. We found the hydrophobic BA glycochenodeoxycholic acid (GCDCA) promotes the replication of the BA-dependent strain GII.3 in jejunal enteroids. Furthermore, we found that inhibition of the G-protein-coupled BA receptor, sphingosine-1-phosphate receptor 2 (S1PR2), by JTE-013, reduced GII.3 infection dose-dependently and inhibited GII.3 cellular uptake in enteroids. Herein, we sought to determine whether S1PR2 is required for other BA-dependent HuNoV strains, the BA-independent GII.4, and whether S1PR2 is required for BA-dependent HuNoV infection in HIEs from other small intestinal segments. We found a second S1PR2 inhibitor, GLPG2938, reduces GII.3 infection dose-dependently, and an S1PR2 agonist (CYM-5520) enhances GII.3 replication in the absence of GCDCA. GII.3 replication also is abrogated in the presence of JTE-013 and CYM-5520. JTE-013 inhibition of S1PR2 in jejunal HIEs reduces GI.1, GII.3, and GII.17 (BA-dependent) but not GII.4 Sydney (BA-independent) infection, providing additional evidence of strain-specific differences in HuNoV infection. Finally, GII.3 infection of duodenal, jejunal, and ileal lines derived from the same individual is reduced with S1PR2 inhibition, indicating a common mechanism of BA-dependent infection among multiple segments of the small intestine. Our results support a model where BA-dependent HuNoVs exploit BA effects on S1PR2 to infect the entire small intestine.IMPORTANCEHuman noroviruses (HuNoVs) are important viral human pathogens that cause both outbreaks and sporadic gastroenteritis. These viruses are diverse, and many strains are capable of infecting humans. Our previous studies have identified strain-specific requirements for hydrophobic bile acids (BAs) to infect intestinal epithelial cells. Moreover, we identified a BA receptor, sphingosine-1-phosphate receptor 2 (S1PR2), required for infection by a BA-dependent strain. To better understand how various HuNoV strains enter and infect the small intestine and the role of S1PR2 in HuNoV infection, we evaluated infection by additional HuNoV strains using an expanded repertoire of intestinal enteroid cell lines. We found that multiple BA-dependent strains, but not a BA-independent strain, all require S1PR2 for infection. In addition, BA-dependent infection requires S1PR2 in multiple segments of the small intestine. Together, these results indicate that S1PR2 has value as a potential therapeutic target for BA-dependent HuNoV infection.

Development of a broad-spectrum therapeutic Fc-nanobody for human noroviruses.

Human norovirus was discovered more than five decades ago and is a widespread cause of outbreaks of acute gastroenteritis. There are no approved vaccines or antivirals currently available. However, norovirus inhibitors, including capsid-specific monoclonal antibodies (Mabs) and nanobodies, have recently shown promising results. Several Mabs and nanobodies were found to inhibit norovirus replication using a human intestinal enteroid (HIE) culture system and/or could block norovirus attachment to histo-blood group antigen (HBGA) co-factors. In our pursuit to develop a single broad-spectrum norovirus therapeutic, we continued our analysis and development of a cross-reactive and HBGA interfering nanobody (NB26). To improve NB26 binding capacity and therapeutic potential, we conjugated NB26 onto a human IgG Fc domain (Fc-NB26). We confirmed that Fc-NB26 cross-reacts with genetically diverse GII genotype capsid protruding (P) domains (GII.8, GII.14, GII.17, GII.24, GII.26, and GII.NA1) using a direct enzyme-linked immunosorbent assay. Furthermore, X-ray crystallography structures of these P domains and structures of other GII genotypes reveal that the NB26 binding site is largely conserved, validating its broad reactivity. We showed that Fc-NB26 has ~100-fold higher affinity toward the norovirus P domain compared to native NB26. We also found that both NB26 and Fc-NB26 neutralize human norovirus replication in the HIE culture system. Furthermore, the mode of inhibition confirmed that like NB26, Fc-NB26 caused norovirus particle disassembly and aggregation. Overall, these new findings demonstrate that structural modifications to nanobodies can improve their therapeutic potential.IMPORTANCEDeveloping vaccines and antivirals against norovirus remains a challenge, mainly due to the constant genetic and antigenic evolution. Moreover, re-infection with genetically related and/or antigenic variants is not uncommon. We further developed our leading norovirus nanobody (NB26) that indirectly interfered with norovirus binding to HBGAs, by converting NB26 into a dimeric Fc-linked Nanobody (Fc-NB26). We found that Fc-NB26 had improved binding affinity and neutralization capacity compared with native NB26. Using X-ray crystallography, we showed this nanobody engaged highly conserved capsid residues among genetically diverse noroviruses. Development of such broadly reactive potent therapeutic nanobodies delivered as a slow-releasing prophylactic could be of exceptional value for norovirus outbreaks, especially for the prevention or treatment of severe acute gastroenteritis in high-risk groups such as the young, elderly, and immunocompromised.

Analysis of Archival Sera From Norovirus-Infected Individuals Demonstrates That Cross-Blocking of Emerging Viruses Is Genotype-Specific.

BACKGROUND: Rapidly evolving RNA viruses, such as human norovirus, generate extraordinary sequence diversity, posing a significant challenge to vaccine design. This diversity, coupled with short-lasting natural immunity, leads to reinfection throughout one's lifetime. How reexposure shapes humoral immunity to future norovirus strains remains incompletely understood. METHODS: We profiled the antibody responses following 2 community gastroenteritis outbreaks with GII.2 and GII.6 noroviruses in 1971. Using diverse virus-like particles (VLPs), enzyme-linked immunosorbent assay (ELISA), and carbohydrate-blocking assays (surrogate for neutralization), we examined the antibody response at acute and convalescent timepoints following GII.6 infection. RESULTS: Convalescent sera displayed strong homologous blocking, demonstrating a 5-fold increase in GII.6 carbohydrate blockade over acute samples, and broad blocking of diverse archival and modern GII.6 noroviruses. Convalescent sera displayed limited carbohydrate blocking of heterotypic VLPs, despite high ELISA binding titers. Select individuals developed broad cross-genotype blockade, but this response was established before the second outbreak. Finally, we applied a novel competitive carbohydrate-blocking assay to demonstrate the epitope specificity and discrete compartments of the neutralizing response. CONCLUSIONS: Our data show that infection generates narrow, focused immunity directed toward the infecting genotype. We did detect broad cross-blocking in specific individuals, but these responses could be attributed to diverse, genotype-specific antibodies predating GII.6 infection.

Differences in Rotavirus Shedding and Duration by Infant Oral Rotavirus Vaccination Status in Dhaka, Bangladesh, 2011-2014.

To evaluate how breakthrough rotavirus disease contributes to transmission, we examined the impact of rotavirus vaccination on fecal shedding and duration of illness. We used multivariable linear regression to analyze rotavirus quantity by RT-qPCR and duration among 184 episodes of rotavirus diarrhea positive by ELISA in the PROVIDE study. Vaccinated children had less fecal viral shedding compared to unvaccinated children (mean difference = -0.59 log copies per gram of stool; 95% confidence interval [CI], -.99 to -.19). Duration of illness was on average 0.47 days (95% CI, -.23 to 1.17 days) shorter among vaccinated children. Rotarix vaccination reduces shedding burden among breakthrough cases of rotavirus gastroenteritis. Clinical Trials Registration . NCT01375647.

Trends for Syndromic Surveillance of Norovirus in Emergency Department Data Based on Chief Complaints.

BACKGROUND: This study compared trends in norovirus cases to determine whether chief complaint-based emergency department (ED) visit data could reflect trends of norovirus in Korea. METHODS: The ED visits from the National Emergency Department Information System database and the weekly reported number of noroviruses from the sentinel surveillance system were collected between August 2017 and December 2020. The correlation between weekly norovirus cases and weekly ED visits considering the chief complaint and discharge diagnosis code was estimated using a 3-week moving average. RESULTS: In total, 6 399 774 patients with chief complaints related to digestive system disease visited an ED. A higher correlation between reported norovirus cases and ED visit with chief complaint of vomiting and discharge diagnosis code of gastroenteritis and colitis of unspecified origin or other and unspecified gastroenteritis and colitis of infectious origin was observed (R = 0.88, P < .0001). The correlation was highest for the age group 0-4 years (R = 0.89, P < .0001). However, no correlation was observed between the reported norovirus cases and the number of ED visits with norovirus identified as a discharge diagnosis code. CONCLUSIONS: ED visit data considering a combination of chief complaints and discharged diagnosis code would be useful for early detection of infectious disease trends.

Wastewater Surveillance for Norovirus, California, USA.

Norovirus is a leading cause of acute gastroenteritis and imposes a substantial disease burden. In California, USA, norovirus surveillance is limited. We evaluated correlations between wastewater norovirus concentrations and available public health surveillance data. Wastewater surveillance for norovirus genotype GII in California provided timely, localized, and actionable data for public health authorities.

Antigenic Characterization of Novel Human Norovirus GII.4 Variants San Francisco 2017 and Hong Kong 2019.

Norovirus is a major cause of acute gastroenteritis; GII.4 is the predominant strain in humans. Recently, 2 new GII.4 variants, Hong Kong 2019 and San Francisco 2017, were reported. Characterization using GII.4 monoclonal antibodies and serum demonstrated different antigenic profiles for the new variants compared with historical variants.

Emerging Variants of Canine Enteric Coronavirus Associated with Outbreaks of Gastroenteric Disease.

A 2022 canine gastroenteritis outbreak in the United Kingdom was associated with circulation of a new canine enteric coronavirus closely related to a 2020 variant with an additional spike gene recombination. The variants are unrelated to canine enteric coronavirus-like viruses associated with human disease but represent a model for coronavirus population adaptation.

Effects of Rotavirus Vaccination Coverage among Infants on Hospital Admission for Gastroenteritis across All Age Groups, Japan, 2011-2019.

We assessed the effect of rotavirus vaccination coverage on the number of inpatients with gastroenteritis of all ages in Japan. We identified patients admitted with all-cause gastroenteritis during 2011-2019 using data from the Diagnosis Procedure Combination system in Japan. We used generalized estimating equations with a Poisson distribution, using hospital codes as a cluster variable to estimate the impact of rotavirus vaccination coverage by prefecture on monthly numbers of inpatients with all-cause gastroenteritis. We analyzed 294,108 hospitalizations across 569 hospitals. Higher rotavirus vaccination coverage was associated with reduced gastroenteritis hospitalizations compared with the reference category of vaccination coverage <40% (e.g., for coverage >80%, adjusted incidence rate ratio was 0.87 [95% CI 0.83-0.90]). Our results show that achieving higher rotavirus vaccination coverage among infants could benefit the entire population by reducing overall hospitalizations for gastroenteritis for all age groups.

Out-of-sync evolutionary patterns and mutual interplay of major and minor capsid proteins in norovirus GII.2.

Human noroviruses are the most common cause of viral gastroenteritis, resulting annually in 219 000 deaths and a societal cost of $60 billion, and no antivirals or vaccines are available. The minor capsid protein may play a significant role in the evolution of norovirus. GII.4 is the predominant genotype of norovirus, and its VP2 undergoes epochal co-evolution with the major capsid protein VP1. Since the sudden emergence of norovirus GII.2[P16] in 2016, it has consistently remained a significant epidemic strain in recent years. In the construction of phylogenetic trees, the phylogenetic trees of VP2 closely parallel those of VP1 due to the shared tree topology of both proteins. To investigate the interaction patterns between the major and minor capsid proteins of norovirus GII.2, we chose five representative strains of GII.2 norovirus and investigated their evolutionary patterns using a yeast two-hybrid experiment. Our study shows VP1-VP2 interaction in GII.2, with critical interaction sites at 167-178 and 184-186 in the highly variable region. In the intra-within GII.2, we observed no temporal co-evolution between VP1 and VP2 of GII.2. Notable distinctions were observed in the interaction intensity of VP2 among inter-genotype (P<0.05), highlighting the divergent evolutionary patterns of VP2 within different norovirus genotypes. In summary, the interactions between VP2 and VP1 of GII.2 norovirus exhibit out-of-sync evolutionary patterns. This study offered valuable insights for further understanding and completing the evolutionary mechanism of norovirus.

Whole-genome analysis of human group A rotaviruses in 1980s Japan and evolutionary assessment of global Wa-like strains across half a century.

Historically, the Wa-like strains of human group A rotavirus (RVA) have been major causes of gastroenteritis. However, since the 2010s, the circulation of non-Wa-like strains has been increasingly reported, indicating a shift in the molecular epidemiology of RVA. Although understanding RVA evolution requires the analysis of both current and historical strains, comprehensive pre-1980's sequencing data are scarce globally. We determined the whole-genome sequences of representative strains from six RVA gastroenteritis outbreaks observed at an infant home in Sapporo, Japan, between 1981 and 1989. These outbreaks were mainly caused by G1 or G3 Wa-like strains, resembling strains from the United States in the 1970s-1980s and from Malawi in the 1990s. Phylogenetic analysis of these infant home strains, together with Wa-like strains collected worldwide from the 1970s to 2020, revealed a notable trend: pre-2010 strains diverged into multiple lineages in many genomic segments, whereas post-2010 strains tended to converge into a single lineage. However, Bayesian skyline plot indicated near-constant effective population sizes from the 1970s to 2020, and selection pressure analysis identified positive selection only at amino acid 75 of NSP2. These results suggest that evidence supporting the influence of rotavirus vaccines, introduced globally since 2006, on Wa-like RVA molecular evolution is lacking at present, and phylogenetic analysis may simply reflect natural fluctuations in RVA molecular evolution. Evaluating the long-term impact of RV vaccines on the molecular evolution of RVA requires sustained surveillance.

Minimal Antigenic Evolution after a Decade of Norovirus GII.4 Sydney_2012 Circulation in Humans.

Norovirus is a major human pathogen that can cause severe gastroenteritis in vulnerable populations. The extensive viral diversity presented by human noroviruses constitutes a major roadblock for the development of effective vaccines. In addition to the large number of genotypes, antigenically distinct variants of GII.4 noroviruses have chronologically emerged over the last 3 decades. The last variant to emerge, Sydney_2012, has been circulating at high incidence worldwide for over a decade. We analyzed 1449 capsid sequences from GII.4 Sydney_2012 viruses to determine genetic changes indicative of antigenic diversification. Phylogenetic analyses show that Sydney_2012 viruses scattered within the tree topology with no single cluster dominating during a given year or geographical location. Fourteen residues presented high variability, 7 of which mapped to 4 antigenic sites. Notably, ~52% of viruses presented mutations at 2 or more antigenic sites. Mutational patterns showed that residues 297 and 372, which map to antigenic site A, changed over time. Virus-like particles (VLPs) developed from wild-type Sydney_2012 viruses and engineered to display all mutations detected at antigenic sites were tested against polyclonal sera and monoclonal antibodies raised against Sydney_2012 and Farmington_Hills_2002 VLPs. Minimal changes in reactivity were detected with polyclonal sera and only 4 MAbs lost binding, with all mapping to antigenic site A. Notably, reversion of residues from Sydney_2012 reconstituted epitopes from ancestral GII.4 variants. Overall, this study demonstrates that, despite circulating for over a decade, Sydney_2012 viruses present minimal antigenic diversification and provides novel insights on the diversification of GII.4 noroviruses that could inform vaccine design. IMPORTANCE GII.4 noroviruses are the major cause of acute gastroenteritis in all age groups. This predominance has been attributed to the continued emergence of phylogenetically discrete variants that escape immune responses to previous infections. The last GII.4 variant to emerge, Sydney_2012, has been circulating at high incidence for over a decade, raising the question of whether this variant is undergoing antigenic diversification without presenting a major distinction at the phylogenetic level. Sequence analyses that include >1400 capsid sequences from GII.4 Sydney_2012 showed changes in 4 out of the 6 major antigenic sites. Notably, while changes were detected in one of the most immunodominant sites over time, these resulted in minimal changes in the antigenic profile of these viruses. This study provides new insights on the mechanism governing the antigenic diversification of GII.4 norovirus that could help in the development of cross-protective vaccines to human noroviruses.

N-Glycosylation of Rotavirus NSP4 Protein Affects Viral Replication and Pathogenesis.

Rotavirus (RV), the most common cause of gastroenteritis in children, carries a high economic and health burden worldwide. RV encodes six structural proteins and six nonstructural proteins (NSPs) that play different roles in viral replication. NSP4, a multifunctional protein involved in various viral replication processes, has two conserved N-glycosylation sites; however, the role of glycans remains elusive. Here, we used recombinant viruses generated by a reverse genetics system to determine the role of NSP4 N-glycosylation during viral replication and pathogenesis. The growth rate of recombinant viruses that lost one glycosylation site was as high as that of the wild-type virus. However, a recombinant virus that lost both glycosylation sites (glycosylation-defective virus) showed attenuated replication in cultured cell lines. Specifically, replications of glycosylation-defective virus in MA104 and HT29 cells were 10- and 100,000-fold lower, respectively, than that of the wild-type, suggesting that N-glycosylation of NSP4 plays a critical role in RV replication. The glycosylation-defective virus showed NSP4 mislocalization, delay of cytosolic Ca2+ elevation, and less viroplasm formation in MA104 cells; however, these impairments were not observed in HT29 cells. Further analysis revealed that assembly of glycosylation-defective virus was severely impaired in HT29 cells but not in MA104 cells, suggesting that RV replication mechanism is highly cell type dependent. In vivo mouse experiments also showed that the glycosylation-defective virus was less pathogenic than the wild-type virus. Taken together, the data suggest that N-glycosylation of NSP4 plays a vital role in viral replication and pathogenicity. IMPORTANCE Rotavirus is the main cause of gastroenteritis in young children and infants worldwide, contributing to 128,500 deaths each year. Here, we used a reverse genetics approach to examine the role of NSP4 N-glycosylation. An N-glycosylation-defective virus showed attenuated and cell-type-dependent replication in vitro. In addition, mice infected with the N-glycosylation-defective virus had less severe diarrhea than mice infected with the wild type. These results suggest that N-glycosylation affects viral replication and pathogenesis. Considering the reduced pathogenicity in vivo and the high propagation rate in MA104 cells, this glycosylation-defective virus could be an ideal live attenuated vaccine candidate.

Human Sapovirus Replication in Human Intestinal Enteroids.

Human sapoviruses (HuSaVs), like human noroviruses (HuNoV), belong to the Caliciviridae family and cause acute gastroenteritis in humans. Since their discovery in 1976, numerous attempts to grow HuSaVs in vitro were unsuccessful until 2020, when these viruses were reported to replicate in a duodenal cancer cell-derived line. Physiological cellular models allowing viral replication are essential to investigate HuSaV biology and replication mechanisms such as genetic susceptibility, restriction factors, and immune responses to infection. In this study, we demonstrate replication of two HuSaV strains in human intestinal enteroids (HIEs) known to support the replication of HuNoV and other human enteric viruses. HuSaVs replicated in differentiated HIEs originating from jejunum, duodenum and ileum, but not from the colon, and bile acids were required. Between 2h and 3 to 6 days postinfection, viral RNA levels increased up from 0.5 to 1.8 log10-fold. Importantly, HuSaVs were able to replicate in HIEs independent of their secretor status and histo-blood group antigen expression. The HIE model supports HuSaV replication and allows a better understanding of host-pathogen mechanisms such as cellular tropism and mechanisms of viral replication. IMPORTANCE Human sapoviruses (HuSaVs) are a frequent but overlooked cause of acute gastroenteritis, especially in children. Little is known about this pathogen, whose successful in vitro cultivation was reported only recently, in a cancer cell-derived line. Here, we assessed the replication of HuSaV in human intestinal enteroids (HIEs), which are nontransformed cultures originally derived from human intestinal stem cells that can be grown in vitro and are known to allow the replication of other enteric viruses. Successful infection of HIEs with two strains belonging to different genotypes of the virus allowed discovery that the tropism of these HuSaVs is restricted to the small intestine, does not occur in the colon, and replication requires bile acid but is independent of the expression of histo-blood group antigens. Thus, HIEs represent a physiologically relevant model to further investigate HuSaV biology and a suitable platform for the future development of vaccines and antivirals.

Unusual G3P[10] bat-like rotavirus strains detected in children with acute gastroenteritis in Thailand.

Rotavirus A (RVA) is the main cause of acute gastroenteritis among children under the age of five globally. The unusual bat-like human RVA strains G3P[10] (RVA/Human-wt/THA/CMH079/05/2005/G3P[10] and RVA/Human-wt/THA/CMH-S015-19/2019/G3P[10]) were detected in children with acute gastroenteritis in 2005 and 2019, respectively, in the same geographical area of Northern Thailand. To elucidate the genetic backgrounds of these unusual or bat-like human RVA strains, the complete genome of these RVA strains was sequenced and phylogenetically analyzed. All eleven genome segments of these G3P[10] strains were genotyped as G3-P[10]-I8-R3-C3-M3-A9-N3-T3-E3-H6, which is closely related to bat G3P[10] RVA strain (RVA/Bat-tc/CHN/MYAS33/2013/G3P[10]) and bat-like human RVA strain (RVA/Human-wt/THA/MS2015-1-0001/2015/G3P[10]). The findings indicate that human G3P[10] RVA strains detected in this study (RVA/Human-wt/THA/CMH079/05/2005/G3P[10] and RVA/Human-wt/THA/CMH-S015-19/2019/G3P[10]) contained all eleven genome segments similar to those of bat RVA strains and appeared to be human RVA strains of bat origin. Phylogenetic analysis revealed that several genome segments of these two RVA strains were also closely related with those of other species in addition to bats and had a zoonotic transmission history. The results of this study supported the roles of interspecies transmission of RVA strains among bats and humans in the natural environment and provided convincing evidence that the evolution of human RVAs was closely interrelated with those of animal RVAs. Continuing surveillance of RVAs in humans and animals is imperative to gain a better understanding of the origin and the evolution of these viruses.

Epidemiology and molecular evolution of GI.1 sapovirus in the recent era.

Sapovirus (SaV) infection is increasing worldwide. Herein, we provided evidence of a significant increase in SaV infection in Japan during 2010-2022, primarily due to the considerable (p = 0.0003) rise of the GI.1 genotype. Furthermore, we found that all major and minor SaV outbreaks in Japan, including the largest SaV outbreak in 2021-2022, were caused by the GI.1 genotype. Therefore, to get insight into the underlying molecular mechanism behind this rising trend of the SaV GI.1 type, we selected 15 SaV GI.1 outbreak strains for complete genome analysis through next-generation sequencing. Phylogenetically, our strains remained clustered in different branches in lineages I and II among the GI.1 genotype. We showed all amino acid (aa) substitutions in different open reading frames (ORFs) in these strains. Importantly, we have demonstrated that the strains involved in the largest SaV outbreak in Japan in 2021-2022 belonged to lineage II and possessed the third ORF. We have identified some unique aa mutations in these major outbreak strains in the NS1 and NS6-NS7 regions that are thought to be associated with viral pathogenicity, cell tropism, and epidemiological competence. Thus, in addition to enriching the database of SaV's complete sequences, this study provides insights into its important mutations.