Human adenovirus (HAdV), especially HAdV species F (HAdV-F) is recognized as a cause of acute gastroenteritis (AGE) worldwide. To assess the global prevalence of HAdV in case-patients of all ages with AGE, we conducted a systematic literature search for studies published in English during 2015-2022. We generated pooled prevalence estimates using generalized linear mixed models. Using data from 147 included articles, the overall pooled prevalence among AGE case-patients of any species of HAdV (pan-HAdV) was 5.8 % and 6.0 % for HAdV-F. The prevalence of HAdV was significantly higher among case-patients < 5 years of age compared with case-patients ≥ 5 years of age (pan-HAdV: 6.6 % vs. 2.0 %, p < 0.0001; HAdV-F: 8.7 % vs. 2.3 %, p = 0.04). Prevalence was significantly higher in high mortality developing countries and lowest in developed countries (pan-HAdV: 9.4 % vs. 4.0 %, p < 0.0001; HAdV-F: 11.6 % vs. 3.2 %, p = 0.0003). Understanding the burden of HAdV-associated AGE may be useful for targeted interventions, including future vaccine development.

Conducting persistence studies of infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on environmental surfaces may require a biosafety level 3 (BSL-3) laboratory. We aimed to compare the environmental persistence of BSL-2 level human coronaviruses (229E, NL63, and OC43) and bovine coronavirus (BoCoV) with three SARS-CoV-2 variants (WA-1, Delta, and Omicron). OC43 (1.8 TCID(50)/mL) and BoCoV (1.0 TCID(50)/mL) had lower detection thresholds in cell culture assays compared to 229E (150 TCID(50)/mL) and NL63 (2,670 TCID(50)/mL) and were used for persistence tests at room temperature. Viable OC43 became undetectable (>5.2log(10)) after 48 hours on stainless steel and plastic coupons but exhibited extended persistence up to 72 hours on touchscreen glass coupons. In contrast, BoCoV remained viable for up to 120 hours with <1.8 log(10) infectivity loss. Both OC43 and BoCoV showed a reduction of >5 log(10) on vinyl coupons after 48 hours. On stainless steel coupons, the viability of all three SARS-CoV-2 variants became undetectable (>2.3 log(10) reduction) after 48 hours, with minor differences in reduction levels at 24 hours, whereas on touchscreen glass coupons, the viable virus could be detected for up to 48 hours for WA-1 and Omicron and 72 hours for the Delta variant. Regardless of coupon or virus type, viral RNA titers increased <4.5 Ct values after 120 hours. Our data demonstrate distinct persistence characteristics between BoCoV and OC43, with neither fully mimicking SARS-CoV-2 variants. This variability along with the impact of surface types on viral persistence underscores the need for caution when using these viruses as surrogates for SARS-CoV-2.IMPORTANCEIn this study, we evaluated three human seasonal coronaviruses (OC43, NL63, and 229E) and one bovine coronavirus (BoCoV) as potential surrogate viruses for SARS-CoV-2. Our data suggest that among the four surrogate viruses tested, OC43 and BoCoV were the most promising candidates due to their assay sensitivity, ease of handling, and high genetic similarity to SARS-CoV-2. However, neither BoCoV nor OC43 fully mimicked the environmental persistence characteristics of SARS-CoV-2 variants highlighting the potential limitations of using surrogate viruses.

BACKGROUND: Noroviruses are the leading cause of acute gastroenteritis worldwide with GII.4 Sydney viruses responsible for the majority of infections until 2023. METHODS: To study the evolutionary dynamics of GII.4 noroviruses in the US (2011-2023), we sequenced and analyzed 406 VP1 and 335 RdRp sequences submitted to CaliciNet. RESULTS: Time-scale analysis showed the average evolutionary rate of GII.4 strains was 5.56 x 10-3 substitutions/site/year and the emergence of a new cluster within GII.4 Sydney every 4 years starting with GII.4 Sydney[P31] from 2011-2015 followed by GII.4 Sydney[P16] from 2016-2020, and the most recent GII.4 Sydney[P16]-2020 from 2021-to date. Since 2017, based on amino acids in VP1, we observed the emergence of three novel GII.4 clusters (GII.4 San Francisco, GII.4 Allegany and GII.4 Wichita). GII.4 Sydney was identified with 4 P-types (P4, P12, P16, and P31). GII.4 San Francisco and GII.4 Allegany had a P31 RdRp, whereas GII.4 Wichita strains had P4. GII.4 Allegany and GII.4 Wichita exhibited major amino acid substitutions in epitopes A-E, G, and H, while GII.4 San Francisco viruses have an alanine insertion in epitope A. Both GII.4 Allegany and GII.4 Wichita VLPs bound porcine gastric mucin at a similar level as GII.4 New Orleans and GII.4 Sydney. However, blocking of binding to VLPs by human serum pools demonstrated their antigenicity was significantly different. CONCLUSION: We identified three new emerging GII.4 noroviruses co-circulating with GII.4 Sydney. Early detection of new strains will aid in tracking their spread and assessing their pandemic potential.

Human norovirus causes more than 700 million illnesses annually. Extensive genetic diversity and a paucity of information on conserved neutralizing epitopes pose major obstacles to the design of broadly protective norovirus immunogens. Here, we used high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS)-driven proteomics to quantitatively characterize the circulating serum IgG repertoire before and after immunization with an experimental monovalent norovirus GII.4 VP1 capsid-encoding adenoviral vaccine. Two participants were specifically selected on the basis of the breadth of serum neutralization responses either across GII.4 variants (participant A) or across GII genotypes (participant B). In participant A, vaccination back-boosted highly abundant serum antibody clonotypes targeting epitopes conserved among rapidly evolving GII.4 variants spanning from a strain identified in 1987 to a strain identified in 2019. In participant B, we identified a recall response consisting of broadly neutralizing monoclonal antibodies with remarkable cross-GII ligand-binding blockade (blocking ≥ seven GII genotypes) and virus neutralization breadth. The cocrystal structure of one of these antibodies, VX22, in complex with the VP1 capsid protruding (P) domain revealed a highly conserved epitope (residues 479 to 484 and 509 to 513) within two lateral loops of the P1 subdomain. Antibody evolutionary trajectory analysis further revealed that VX22 had originally evolved from an early heterologous infection, likely by a GII.12 strain. Together, our study demonstrates that norovirus human monoclonal antibodies with broad GII.4 potency and cross-GII breadth can be boosted in serum after immunization with an adenoviral vector-based vaccine, findings that may guide the design of immunogens for broadly protective norovirus vaccines.

Globally, human norovirus (HuNoV) is the leading cause of foodborne illnesses. Norovirus transmission to fresh produce can occur via several sources, including contaminated irrigation water. HuNoV RNA has been detected in freshwater resources, but knowledge about virus infectivity is limited due to a historical lack of a HuNoV cell culture. Recently, HuNoV was shown to replicate in human intestinal enteroids (HIE). The objective of this study was to use HIE to evaluate the persistence of infectious HuNoV in raw (i.e. biologically active) surface freshwater. The virus was spiked into freshwater microcosms sampled from three freshwater ponds and then incubated inside an environmental chamber at 20-15 °C and 50-80 % relative humidity (day-night) and 12 h photoperiod. The water was tested for infectious HuNoV, intact HuNoV capsids, indigenous bacteria, and other water quality parameters over a period of 2 weeks. The persistence of infectious HuNoV in the three freshwater microcosms ranged from ≤1 day to ≥7 days. Decay rates for RNA from intact HuNoV capsids ranged from 0.04 to 0.54/day, predicting a 4.2 to 57.5 days, respectively for 1 log reduction. The intact virus showed a significant negative and positive linear relationship with indigenous bacteria and dissolved oxygen, respectively. Using multiple logistic regression, HuNoV RNA >4.4 log genomic equivalent/ml (Cycle threshold values <32) predicted higher probability of detecting infectious HuNoV in contaminated raw freshwater using HIE. Overall, our results provide valuable insights for enhancing quantitative microbial risk assessment models for pre-harvest agricultural water to understand the public health risks associated with the detection of HuNoV RNA in freshwater.

Norovirus is the leading cause of acute gastroenteritis (AGE) globally. Few longitudinal studies have assessed norovirus-associated AGE incidence across age groups in community settings in Latin America. During April 2015-April 2019, active surveillance for AGE among community members of all ages was conducted through household visits two to three times per week in San Jeronimo, Cusco, Peru. An asymptomatic control household was selected for every fifth AGE case. Stool specimens were collected from AGE cases, asymptomatic household members, and control household members, and they were tested for norovirus using real-time reverse transcriptase polymerase chain reaction. Data on illness characteristics were collected from AGE cases during a 15-day follow-up period. Annual means of 247 households and 1,555 participants were enrolled during each April-March surveillance year, accounting for 4,176 person-years (PY) of observation. Of 1,099 AGE events reported, 1,014 stool specimens were tested, and 186 (18%) were norovirus positive. Norovirus AGE incidence was 4.4/100 PY (95% CI: 3.9-5.1); incidence was highest among those younger than 2 years old (60.9/100 PY; 95% CI: 46.8-79.4). Among 672 stool specimens from asymptomatic controls, 56 (8%) tested positive for norovirus. Odds of norovirus detection were significantly higher among cases compared with controls (odds ratio: 2.2; 95% CI: 1.6-3.1). Age-stratified norovirus incidence in this periurban community aligns with previously published estimates and was highest among those younger than 2 years old. Establishing baseline norovirus incidence in specific communities is crucial to identify target populations and assess effectiveness of future interventions, such as vaccines.

We investigated a suspected norovirus outbreak associated with a wedding reception in Wisconsin in May 2015. Fifty-six of 106 (53%) wedding attendees were interviewed and 23 (41%) reported symptoms consistent with norovirus infection. A retrospective cohort study identified fruit salad as the likely vehicle of infection (risk ratio 3.2, 95% confidence interval 1.1- 8.3). Norovirus was detected by real-time reverse transcription polymerase chain reaction (RT-qPCR) in stool specimens collected from four attendees and one food handler and in 12 leftover fruit salad samples from both an opened and a sealed container. Norovirus-positive clinical samples (n=4) were genotyped as GII.4 Sydney and norovirus-positive fruit salad samples (n=2) confirmed the presence of GII.4 norovirus by Sanger sequencing with 98% nucleotide (n=236) similarity in 5' end of ORF2 between fruit salad and clinical specimens. In conclusion, this comprehensive norovirus outbreak investigation combined epidemiologic, virologic, and environmental findings to traceback the contaminated food as the source of the outbreak.

We report an increase in GII.17 norovirus outbreaks and sporadic infections of acute gastroenteritis in Austria, Germany, France, Ireland, the Netherlands, England and the United States during the 2023/24 season. A decrease in GII.4 coincided with GII.17 prevalence increasing to between 17% and 64% of all GII detections. Overall, 84% of the GII.17 strains clustered closely with strains first reported in Romania in 2021 and two new sub-lineages were identified. Norovirus surveillance and molecular characterisation should be prioritised this winter.

BACKGROUND: Astrovirus is a leading cause of acute gastroenteritis in children worldwide. However, few prospective studies have analyzed astrovirus in community-dwelling pediatric populations in low- and middle-income countries. METHODS: We assessed the incidence, risk factors, clinical characteristics, genotypes, viral coinfections, and time distribution of astrovirus gastroenteritis in 443 healthy Nicaraguan children born in 2017 to 2018 who were followed for 36 months. Children were recruited from hospitals and birth records in an economically diverse neighborhood of León city. Astrovirus-positive episodes and genotypes were identified from stool with reverse transcription quantitative polymerase chain reaction and Sanger sequencing. RESULTS: Of 1708 total specimens tested, 80 children (18%) experienced at least 1 astrovirus episode, and 9 experienced repeat episodes, mostly during the rainy season (May-October). Initial astrovirus episodes were not associated with a lowered risk against future episodes. In exploratory analyses, home toilets were associated with a lower risk of future astrovirus episodes (hazard ratio, 0.19; 95% CI, .04-.91). Human astrovirus 5 episodes, representing 15% of all typed episodes, were associated with longer diarrhea and more symptomatic rotavirus coinfections. CONCLUSIONS: Astrovirus was a common cause of gastroenteritis in this cohort, and future studies should clarify the role of astrovirus genotype in clinical infection severity.

mRNA-based COVID-19 vaccines have played a critical role in reducing severe outcomes of COVID-19. Humoral immune responses against SARS-CoV-2 after vaccination have been extensively studied in blood; however, limited information is available on the presence and duration of SARS-CoV-2 specific antibodies in saliva and other mucosal fluids. Saliva offers a non-invasive sampling method that may also provide a better understanding of mucosal immunity at sites where the virus enters the body. Our objective was to evaluate the salivary immune response after vaccination with the COVID-19 Moderna mRNA-1273 vaccine. Two hundred three staff members of the U.S. Centers for Disease Control and Prevention were enrolled prior to receiving their first dose of the mRNA-1273 vaccine. Participants were asked to self-collect 6 saliva specimens at days 0 (prior to first dose), 14, 28 (prior to second dose), 42, and 56 using a SalivaBio saliva collection device. Saliva specimens were tested for anti-spike protein SARS-CoV-2 specific IgA and IgG enzyme immunoassays. Overall, SARS-CoV-2-specific salivary IgA titers peaked 2 weeks after each vaccine dose, followed by a sharp decrease during the following weeks. In contrast to IgA titers, IgG antibody titers increased substantially 2 weeks after the first vaccine dose, peaked 2 weeks after the second dose and persisted at an elevated level until at least 8 weeks after the first vaccine dose. Additionally, no significant differences in IgA/IgG titers were observed based on age, sex, or race/ethnicity. All participants mounted salivary IgA and IgG immune responses against SARS-CoV-2 after receiving the mRNA-1273 COVID-19 vaccine. Because of the limited follow-up time for this study, more data are needed to assess the antibody levels beyond 2 months after the first dose. Our results confirm the potential utility of saliva in assessing immune responses elicited by immunization and possibly by infection.

BACKGROUND: Sapovirus is an important cause of acute gastroenteritis in childhood. While vaccines against sapovirus may reduce gastroenteritis burden, a major challenge to their development is a lack of information about natural immunity. METHODS: We measured sapovirus-specific IgG in serum collected, between 2017 and 2020, of mothers soon after delivery and at 6 time points in Nicaraguan children until 3 years of age (n=112 dyads) using virus-like particles representing three sapovirus genotypes (GI.1, GI.2, GV.1). RESULTS: Sixteen (14.3%) of the 112 children experienced at least one sapovirus gastroenteritis episode, of which GI.1 was the most common genotype. Seroconversion to GI.1 and GI.2 was most common between 5 and 12 months of age, while seroconversion to GV.1 peaked at 18 to 24 months of age. All children who experienced sapovirus GI.1 gastroenteritis seroconverted and developed genotype-specific IgG. The impact of sapovirus exposure on population immunity was determined using antigenic cartography: newborns share their mothers' broadly binding IgG responses, which declined at 5 months of age and then increased as infants experienced natural sapovirus infections. CONCLUSION: By tracking humoral immunity to sapovirus over the first 3 years of life, this study provides important insights for the design and timing of future pediatric sapovirus vaccines.

BACKGROUND: Most U.S. acute gastroenteritis (AGE) episodes in children are attributed to norovirus, whereas very little information is available on adenovirus 40/41 (AdV40/41), astrovirus or sapovirus. The New Vaccine Surveillance Network (NVSN) conducted prospective, active, population-based AGE surveillance in young children. METHODS: We tested and typed stool specimens collected between December 2011 to June 2016 from one NVSN site in Kansas City for the three viruses, and calculated hospitalization and emergency department (ED) detection rate. RESULTS: Of 3,205 collected specimens, 2,453 (76.5%) were from AGE patients (339 inpatients and 2,114 ED patients) and 752 (23.5%) were from healthy controls (HC). In AGE patients, astrovirus was detected in 94 (3.8%), sapovirus in 252 (10.3%) and AdV40/41 in 101 (4.5%) of 2249 patients. In HC, astrovirus was detected in 13 (1.7%) and sapovirus in 15 (2.0%) specimens. Astrovirus type 1 (37.7%) and genogroup I sapoviruses (59.3%) were most prevalent.Hospitalization rates were 5 (AdV40/41), 4 (astrovirus) and 8 (sapovirus) per 100,000 children <11 years old, whereas ED rates were 2.4 (AdV40/41), 1.9 (astrovirus) and 5.3 (sapovirus) per 1000 children <5 years old. CONCLUSIONS: Overall, AdV40/41, astrovirus, and sapovirus were detected in 18.6% of AGE in a large pediatric hospital in Kansas City.

CrAssphages are human gut bacteriophages with potential use as an indicator of human fecal contamination in water and other environmental systems. We determined the prevalence and abundance of crAssphages in water, food, and fecal samples and compared these estimates with the prevalence of norovirus. Samples were tested using two crAssphage-specific qPCR assays (CPQ056 and TN201-203) and for norovirus using TaqMan realtime RT-PCR. CrAssphage was detected in 40% of human fecal specimens, 61% of irrigation water samples, 58.5% of stream water samples, and 68.5% of fresh leafy greens samples. Interestingly, across all sample categories, crAssphage concentrations were 2-3 log10 higher than norovirus concentrations. The correlation of detection of crAssphage and norovirus was significant for the irrigation water samples (r = 0.74, p = 7.4e-06). Sequences obtained from crAssphage positive samples from human fecal and stream water samples phylogenetically clustered with genotype I crAssphages, whereas sequences derived from irrigation water samples clustered differently from other genotypes. Our data show that crAssphages were prevalent in norovirus-positive water samples and in fresh leafy green samples, there was a strong correlation between the presence of crAssphage and norovirus. CrAssphage genomic copies were consistently higher than norovirus copies in all sample types. Overall, our findings suggest that crAssphages could be used as reliable indicators to monitor fecal-borne virus contamination within the food safety chain.

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.

AIMS: This study aimed to compare the heat inactivation kinetics of viable human norovirus with the surrogate, MS2 bacteriophage as well as assess the decay of the RNA signal. METHODS AND RESULTS: Human intestinal enteroids (HIEs) were used to analyze the heat inactivation kinetics of viable human norovirus compared to the surrogate MS2 bacteriophage, which was cultured using a plaque assay. Norovirus decay rates were 0.22 min-1, 0.68 min-1, and 1.11 min-1 for 50°C, 60°C, and 70°C, respectively, and MS2 bacteriophage decay rates were 0.0065 min-1, 0.045 min-1, and 0.16 min-1 for 50°C, 60°C, and 70°C, respectively. Norovirus had significantly higher decay rates than MS2 bacteriophage at all tested temperatures (P = 0.002-0.007). No decrease of RNA titers as measured by reverse transcription-PCR for both human norovirus and MS2 bacteriophage over time was observed, indicating molecular methods do not accurately depict viable human norovirus after heat inactivation and treatment efficiency is underestimated. CONCLUSIONS: Overall, our data demonstrates that MS2 bacteriophage is a conservative surrogate to measure heat inactivation and potentially overestimates the infectious risk of norovirus. Furthermore, this study corroborates that measuring viral RNA titers, as evaluated by PCR methods, does not correlate with the persistence of viable norovirus under heat inactivation.

Acute gastroenteritis (AGE) is a leading cause of morbidity and mortality worldwide across all age groups that disproportionally affects young children in low- and middle-income countries and immunocompromised patients in high-income countries. Regional outbreaks of AGE are typically detected by traditional microbiological detection methods that target limited organisms and are associated with low sensitivity and lengthy time-to-results. Combined, these may result in repeat testing, imprecise or delayed treatment, and delayed recognition of outbreaks. We conducted a multi-site prospective study comparing the BioCode Gastrointestinal Pathogen Panel (BioCode GPP) for the detection of 17 common bacterial, viral, and protozoan causes of gastroenteritis with reference methods, including stool culture, enzyme immunoassays, pathogen-specific PCR assays, and sequencing. One thousand five hundred fifty-eight residual, de-identified stool samples (unpreserved stool and stool in Cary-Blair transport medium) were enrolled and tested for 11 bacterial, 3 viral, and 3 protozoan pathogens. BioCode GPP and reference methods were positive for 392 (25.2%) and 283 (18.2%) samples, respectively (P < 0.0001). In this study, the BioCode GPP and reference methods detected 69 and 65 specimens positive for Clostridioides difficile, 51 and 48 for enteroaggregative Escherichia coli, 33 and 27 for enterotoxigenic E. coli, 50 and 47 for norovirus GI/GII, and 30 and 22 for rotavirus A, respectively. The BioCode GPP showed good positive and negative agreements for each pathogen ranging from 89.5% to 100%, with overall sensitivity and specificity of 96.1% and 99.7%, post adjudication. The BioCode GPP detected >1 pathogens in 49 samples, representing 12.5% of the total 392 positive specimens.IMPORTANCEThis study highlights performance of a novel technology for timely and accurate detection and differentiation of 17 common bacterial, viral, and protozoan causes of gastroenteritis. Utilizing molecular tests such as the BioCode Gastrointestinal Pathogen Panel may improve the detection of gastrointestinal pathogens and provide actionable results, particularly for patient populations at most risk.

Viral metagenomics sequencing of fecal samples from outbreaks of acute gastroenteritis from the US revealed the presence of small circular ssDNA viral genomes encoding a replication initiator protein (Rep). Viral genomes were ∼2.5 kb in length, with bi-directionally oriented Rep and capsid (Cap) encoding genes and a stem loop structure downstream of Rep. Several genomes showed evidence of recombination. By digital screening of an in-house virome database (1.04 billion reads) using BLAST, we identified closely related sequences from cases of unexplained diarrhea in France. Deep sequencing and PCR detected such genomes in 7 of 25 US (28 percent) and 14 of 21 French outbreaks (67 percent). One of eighty-five sporadic diarrhea cases in the Gambia was positive by PCR. Twenty-two complete genomes were characterized showing that viruses from patients in the same outbreaks were closely related suggesting common origins. Similar genomes were also characterized from the stools of captive chimpanzees, a gorilla, a black howler monkey, and a lemur that were more diverse than the human stool-associated genomes. The name smacovirus is proposed for this monophyletic viral clade. Possible tropism include mammalian enteric cells or ingested food components such as infected plants. No evidence of viral amplification was found in immunodeficient mice orally inoculated with smacovirus-positive stool supernatants. A role for smacoviruses in diarrhea, if any, remains to be demonstrated.

After the introduction of the rotavirus vaccine into the Universal Immunization Program in India in 2016, relatively few studies have assessed the prevalence and epidemiological patterns of acute gastroenteritis (AGE) among hospitalized children ≤5 years of age. We used a uniform protocol to recruit children with AGE as well as standardized testing and typing protocols. Stool specimens from children with AGE younger than 5 years of age admitted to six hospitals in three cities in India were collected from January 2017 through December 2019. Norovirus was detected by real-time reverse transcription-polymerase chain reaction (RT-qPCR) followed by typing positive specimens by conventional RT-PCR and Sanger sequencing. Norovirus was detected in 322 (14.8%) of 2182 specimens with the highest rate in 2018 (17.6%, 146/829), followed by 2019 (14.4%, 122/849) and 2017 (10.7%, 54/504). Rotavirus vaccine status was known for 91.6% of the children of which 70.4% were vaccinated and 29.6% not. Norovirus positivity in rotavirus-vaccinated children was 16.3% and 12% in unvaccinated children. GII.4 Sydney[P16] (39.3%), GII.4 Sydney[P31] (18.7%), GII.2[P16] (10%), GI.3[P13] (6.8%), GII.3[P16] (5.9%), and GII.13[P16] (5%) accounted for 85.8% (188/219) of the typed strains. Our data highlight the importance of norovirus in Indian children hospitalized with AGE.

We detected a novel GII.4 variant with an amino acid insertion at the start of epitope A in viral protein 1 of noroviruses from the United States, Gabon, South Africa, and the United Kingdom collected during 2017-2022. Early identification of GII.4 variants is crucial for assessing pandemic potential and informing vaccine development.

Norovirus is attributed to nearly 1 out of every 5 episodes of diarrheal disease globally and is estimated to cause approximately 200,000 deaths annually worldwide, with 70,000 or more among children in developing countries. Noroviruses remain a leading cause of sporadic disease and outbreaks of acute gastroenteritis even in industrialized settings, highlighting that improved hygiene and sanitation alone may not be fully effective in controlling norovirus. Strengths in global progress towards a Norovirus vaccine include a diverse though not deep pipeline which includes multiple approaches, including some with proven technology platforms (e.g., VLP-based HPV vaccines). However, several gaps in knowledge persist, including a fulsome mechanistic understanding of how the virus attaches to human host cells, internalizes, and induces disease. © 2023 The Author(s)

BACKGROUND: Norovirus is a major cause of endemic acute gastroenteritis (AGE) worldwide. We described the epidemiology, risk factors, and genotypic distribution of noroviruses among hospitalized patients of all ages in Bangladesh. METHODS: From March 2018 to October 2021, 1250 AGE case patients and controls (age, sex, season, and site matched) were enrolled at 10 hospitals. Demographic and clinical information was collected; real-time reverse-transcriptase polymerase chain reaction (RT-PCR) used to test stool specimens, and positive samples were genotyped. RESULTS: Norovirus was detected in 9% of cases (111 of 1250) and 15% (182 of 1250) of controls. Eighty-two percent of norovirus-positive cases were in children <5 years old. Norovirus-positive AGE hospitalizations occurred year-round, with peaks in April and October. Risk factors for norovirus included age <5 years (adjusted odds ratio, 3.1 [95% confidence interval, 1.9-5.2]) and exposure to a patient with AGE in the 10 days before enrollment (3.8 [1.9-7.2]). GII.3[P16] and GII.4 Sydney[P16] were the predominant genotypes. CONCLUSIONS: We highlight the burden of norovirus in hospital settings. Young age and recent exposure to a patient with AGE were risk factors for norovirus. A high prevalence of norovirus among controls might represent asymptomatic reinfections or prolonged shedding from a previous infection; carefully designed longitudinal studies are needed to improve our understanding of norovirus infections in Bangladesh.

BACKGROUND: Adenovirus is a known cause of hepatitis in immunocompromised children, but not in immunocompetent children. In April, 2022, following multiple reports of hepatitis of unknown aetiology and adenovirus viraemia in immunocompetent children in the USA and UK, the US Centers for Disease Control and Prevention (CDC) and jurisdictional health departments initiated national surveillance of paediatric acute hepatitis of unknown aetiology. We aimed to describe the clinical and epidemiological characteristics of children identified with hepatitis of unknown aetiology between Oct 1, 2021, and Sept 30, 2022, in the USA and to compare characteristics of those who tested positive for adenovirus with those who tested negative. METHODS: In this national surveillance investigation in the USA, children were identified for investigation if they were younger than 10 years with elevated liver transaminases (>500 U/L) who had an unknown cause for their hepatitis and onset on or after Oct 1, 2021. We reviewed medical chart abstractions, which included data on demographics, underlying health conditions, signs and symptoms of illness, laboratory results, vaccination history, radiological and liver pathology findings, diagnoses and treatment received, and outcomes. Caregiver interviews were done to obtain information on symptoms and health-care utilisation for the hepatitis illness, medical history, illness in close contacts or at school or daycare, diet, travel, and other potential exposures. Blood, stool, respiratory, and tissue specimens were evaluated according to clinician discretion and available specimens were submitted to CDC for additional laboratory testing or pathology evaluation. FINDINGS: Surveillance identified 377 patients from 45 US jurisdictions with hepatitis of unknown aetiology with onset from Oct 1, 2021, to Sept 30, 2022. The median age of patients was 2·8 years (IQR 1·2-5·0) and 192 (51%) were male, 184 (49%) were female, and one patient had sex unknown. Only 22 (6%) patients had a notable predisposing underlying condition. 347 patients (92%) were admitted to hospital, 21 (6%) subsequently received a liver transplant, and nine (2%) died. Among the 318 patients without notable underlying conditions, 275 were tested for adenovirus. Of these 116 (42%) had at least one positive specimen, and species F type 41 was the most frequent type identified (19 [73%] of 26 typed specimens were HAdV-41). Proportions of patients who had acute liver failure, received a liver transplant, and died were similar between those who tested positive for adenovirus compared with those who tested negative. Adenovirus species F was detected by polymerase chain reaction in nine pathology liver evaluations, but not by immunohistochemistry in seven of the nine with adequate liver tissue available. Interviews with caregivers yielded no common exposures. INTERPRETATION: Adenovirus, alone or in combination with other factors, might play a potential role in acute hepatitis among immunocompetent children identified in this investigation, but the pathophysiologic mechanism of liver injury is unclear. To inform both prevention and intervention measures, more research is warranted to determine if and how adenovirus might contribute to hepatitis risk and the potential roles of other pathogens and host factors. FUNDING: None.

While the COVID-19 pandemic has had a detrimental impact on many businesses worldwide, essential businesses, such as grocery stores, continued to operate despite potential disease transmission. Although the principal mode by which people are infected with SARS-CoV-2, the virus that causes COVID-19, is through exposure to respiratory droplets and very small particles carrying infectious virus, contaminated surfaces might play a role in transmission. We collected swab samples from frequently touched surfaces, including grocery carts, touchscreen monitors, credit card keypads, pharmacy counters, self-service food utensils, and refrigerator and freezer handles, in two metro-Atlanta grocery stores over the course of two sampling events in March 2021. Of the 260 swab samples collected, 6 (2.3%) samples were positive for SARS-CoV-2 RNA by reverse transcriptase quantitative polymerase chain reaction. Positive samples were collected from pharmacy (12.0% [3/25] samples), refrigerator/freezer aisles (2.5% [1/39] samples), and self-service food court (5.0% [2/40] samples) areas. Table/counter edge and underside surfaces represented 33% (2/6) of positive samples. These data suggest that risk of exposure to SARS-CoV-2 from frequently touched surfaces in grocery store settings is likely low; however, more frequent cleaning of surfaces in pharmacy and self-service food courts might be warranted.

Genogroup II (GII) noroviruses are a major cause of diarrheal disease burden in children in both high- and low-income countries. GII.17 noroviruses are composed of distinct genetic clusters (I, II, IIIa, and IIIb) and have shown potential for replacing historically more prevalent GII.4 strains, but the serological basis for GII.17 antigenic diversity has not been studied in children. Utilizing samples from a birth cohort, we investigated antibody and B-cell responses to GII.17 cluster variants in confirmed GII.17 infections in young children as well as demonstrated that the distinct genetic clusters co-circulate. Polyclonal serum antibodies bound multiple clusters but showed cluster-specific blockade activity in a surrogate virus neutralization assay. Antibodies secreted by immortalized memory B cells (MBCs) from an infant GII.17 case were highly specific to GII.17 and exhibited blockade activity against this genotype. We isolated an MBC-derived GII.17-specific Immunoglobulin A (IgA) monoclonal antibody called NVA.1 that potently and selectively blocked GII.17 cluster IIIb and recognized an epitope targeted in serum from cluster IIIb-infected children. These data indicate that multiple antigenically distinct GII.17 variants co-circulate in young children, suggesting retention of cluster diversity alongside potential for immune escape given the existence of antibody-defined cluster-specific epitopes elicited during infection.

The control of pandemic pathogens depends on early prediction of pandemic variants and, more generally, understanding origins of such variants and factors that drive their global spread. This is especially important for GII.4 norovirus, where vaccines under development offer promise to prevent hundreds of millions of annual gastroenteritis cases. Previous studies have suggested that new GII.4 pandemic viruses evolve from previous pandemic variants through substitutions in the antigenic region of the VP1 protein that enable evasion of host population immunity, leading to global spread. In contrast, we show here that the acquisition of new genetic and antigenic characteristics is not the proximal driver of new pandemics. Instead, pandemic GII.4 viruses circulate undetected for years before causing a new pandemic, during which time they diversify and spread over wide geographical areas. Serological data demonstrate that by 2003, some nine years before it emerged as a new pandemic, the ancestral 2012 pandemic strain had already acquired the antigenic characteristics that allowed it to evade prevailing population immunity against the previous 2009 pandemic variant. These results provide strong evidence that viral genetic changes are necessary but not sufficient for GII.4 pandemic spread. Instead, we suggest that it is changes in host population immunity that enable pandemic spread of an antigenically-preadapted GII.4 variant. These results indicate that predicting future GII.4 pandemic variants will require surveillance of currently unsampled reservoir populations. Furthermore, a broadly acting GII.4 vaccine will be critical to prevent future pandemics.Significance Norovirus pandemics and their associated public health and economic costs could be prevented by effective vaccines. However, vaccine development and distribution will require identification of the sources and drivers of new pandemics. We here use phylogenetics and serological experiments to develop and test a new hypothesis of pandemic norovirus emergence. We find that pandemic noroviruses preadapt, diversify and spread worldwide years prior to emergence, strongly indicating that genetic changes are necessary but not sufficient to drive a new pandemic. We instead suggest that changes in population immunity enable pandemic emergence of a pre-adapted low-level variant. These findings indicate that prediction of new pandemics will require surveillance of under-sampled virus reservoirs and that norovirus vaccines will need to elicit broad immunity.

Little is known about enteropathogen seroepidemiology among children in low-resource settings. We measured serological IgG responses to eight enteropathogens (Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica, Salmonella enterica, enterotoxigenic Escherichia coli, Vibrio cholerae, Campylobacter jejuni, norovirus) in cohorts from Haiti, Kenya, and Tanzania. We studied antibody dynamics and force of infection across pathogens and cohorts. Enteropathogens shared common seroepidemiologic features that enabled between-pathogen comparisons of transmission. Overall, exposure was intense: for most pathogens the window of primary infection was &lt;3 years old; for highest transmission pathogens primary infection occurred within the first year. Longitudinal profiles revealed significant IgG boosting and waning above seropositivity cutoffs, underscoring the value of longitudinal designs to estimate force of infection. Seroprevalence and force of infection were rank-preserving across pathogens, illustrating the measures provide similar information about transmission heterogeneity. Our findings suggest multiplex antibody assays are a promising approach to measure population-level enteropathogen transmission in serologic surveillance.

Next-generation sequencing is a powerful tool for virological surveillance. While Illumina® and Ion Torrent® sequencing platforms are used extensively for generating viral RNA genome sequences, there is limited data comparing different platforms. We evaluated the Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5 platforms using a panel of sixteen specimens containing picornaviruses and human caliciviruses (noroviruses and sapoviruses). The specimens were processed, using combinations of three library preparation and five sequencing kits, to assess the quality and completeness of assembled viral genomes, and an estimation of cost per sample to generate the data was calculated. The choice of library preparation kit and sequencing platform was found to impact the breadth of genome coverage and accuracy of consensus viral genomes. The Ion Torrent S5 outperformed the older Ion Torrent PGM platform in data quality and cost, and generated the highest proportion of reads for enterovirus D68 samples. However, indels at homopolymer regions impacted the accuracy of consensus genome sequences. For lower throughput sequencing runs (i.e., Ion Torrent 510 or Illumina MiSeq Nano V2), the cost per sample was lower on the MiSeq platform, whereas with higher throughput runs (Ion Torrent 530 or Illumina MiSeq V2) the cost per sample was comparable. These findings suggest that the Ion Torrent S5 and Illumina MiSeq platforms are both viable options for genomic sequencing of RNA viruses, each with specific advantages and tradeoffs.

Rotaviruses (RVs) preferentially replicate in the small intestine, frequently cause severe diarrheal disease, and following enteric infection generally induce variable levels of protective systemic and mucosal immune responses in humans and other animals. Rhesus rotavirus (RRV) is a simian RV that was previously used as a human RV vaccine and has been extensively studied in mice. Although RRV replicates poorly in the suckling mouse intestine, infection induces a robust and protective antibody response. The recent availability of plasmid-based RV reverse genetics systems has enabled the generation of recombinant RVs expressing foreign proteins. However, recombinant RVs have not yet been experimentally tested as potential vaccine vectors to immunize against other gastrointestinal pathogens in vivo. This is a missed opportunity because several live-attenuated RV vaccines are already widely administered to infants and young children worldwide. To explore the feasibility of using RV as a dual vaccine vector, we rescued a replication-competent recombinant RRV harboring bicistronic gene segment 7 that encodes both the native RV NSP3 protein and a human norovirus (HuNoV) VP1 protein from the predominant genotype GII.4 (rRRV-HuNoV-VP1). The rRRV-HuNoV-VP1 expressed HuNoV VP1 in infected cells in vitro and importantly, elicited both systemic and local antibody responses to HuNoV following oral infection of suckling mice. Serum IgG and fecal IgA from infected suckling mice bound to and neutralized both RV and HuNoV. These findings have encouraging practical implications for the design of RV-based next-generation multivalent enteric vaccines to target HuNoV and other human enteric pathogens while providing immunity to RV. Copyright The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.

Oral fluids offer a non-invasive sampling method for the detection of antibodies. Quantification of IgA and IgG antibodies in saliva allows studies of the mucosal and systemic immune response after natural infection or vaccination. We developed and validated an enzyme immunoassay (EIA) to detect and quantify salivary IgA and IgG antibodies against the prefusion-stabilized form of the SARS-CoV-2 spike protein. Normalization against total antibody isotype was performed to account for specimen differences, such as collection time and sample volume. Saliva samples collected from 187 SARS-CoV-2 confirmed cases enrolled in 2 cohorts and 373 pre-pandemic saliva samples were tested. The sensitivity of both EIAs was high (IgA: 95.5%; IgG: 89.7%) without compromising specificity (IgA: 99%; IgG: 97%). No cross reactivity with seasonal coronaviruses was observed. The limit of detection for SARS-CoV-2 salivary IgA and IgG assays were 1.98 ng/mL and 0.30 ng/mL, respectively. Salivary IgA and IgG antibodies were detected earlier in patients with mild COVID-19 symptoms than in severe cases. However, severe cases showed higher salivary antibody titers than those with a mild infection. Salivary IgA titers quickly decreased after 6 weeks in mild cases but remained detectable until at least week 10 in severe cases. Salivary IgG titers remained high for all patients, regardless of disease severity. In conclusion, EIAs for both IgA and IgG had high specificity and sensitivity for the confirmation of current or recent SARS-CoV-2 infections and evaluation of the IgA and IgG immune response.

CaliciNet China, a network of provincial, county, and city laboratories coordinated by the Chinese Centers for Disease Control and Prevention, was launched in October 2016 to monitor the epidemiology and genotype distribution of norovirus outbreaks in China. During October 2016-September 2018, a total of 556 norovirus outbreaks were reported, and positive fecal samples from 470 (84.5%) outbreaks were genotyped. Most of these outbreaks were associated with person-to-person transmission (95.1%), occurred in childcare centers or schools (78.2%), and were reported during November-March of each year (63.5%). During the 2-year study period, 81.2% of all norovirus outbreaks were typed as GII.2[P16]. In China, most norovirus outbreaks are reported by childcare centers or schools; GII.2[P16] is the predominant genotype. Ongoing surveillance by CaliciNet China will provide information about the evolving norovirus genotype distribution and outbreak characteristics important for the development of effective interventions, including vaccines.