Human intestinal enteroids platform to assess the infectivity of gastroenteritis viruses in wastewater.

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.

Infant antibody and B-cell responses following confirmed pediatric GII.17 norovirus infections functionally distinguish GII.17 genetic clusters.

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.

Mucosal and systemic neutralizing antibodies to norovirus induced in infant mice orally inoculated with recombinant rotaviruses.

Rotaviruses (RVs) preferentially replicate in the small intestine and frequently cause severe diarrheal disease, and the following enteric infection generally induces 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 only-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 newly available 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 replication-competent recombinant RRVs harboring bicistronic gene segment 7 that encodes the native RV nonstructural protein 3 (NSP3) protein and a human norovirus (HuNoV) VP1 protein or P domain from the predominant genotype GII.4. The rescued viruses expressed HuNoV VP1 or P protein in infected cells in vitro and elicited systemic and local antibody responses to HuNoV and RRV following oral infection of suckling mice. Serum IgG and fecal IgA from infected suckling mice bound to and neutralized both RRV 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.

Persistence of Human Norovirus (GII) in Surface Water: Decay Rate Constants and Inactivation Mechanisms.

Human norovirus (HuNoV) is an important cause of acute gastroenteritis and can be transmitted by water exposures, but its persistence in water is not well understood. Loss of HuNoV infectivity in surface water was compared with persistence of intact HuNoV capsids and genome segments. Surface water from a freshwater creek was filter-sterilized, inoculated with HuNoV (GII.4) purified from stool, and incubated at 15 or 20 °C. We measured HuNoV infectivity via the human intestinal enteroid system and HuNoV persistence via reverse transcription-quantitative polymerase chain reaction assays without (genome segment persistence) or with (intact viral capsid persistence) enzymatic pretreatment to digest naked RNA. For infectious HuNoV, results ranged from no significant decay to a decay rate constant ("k") of 2.2 day-1. In one creek water sample, genome damage was likely a dominant inactivation mechanism. In other samples from the same creek, loss of HuNoV infectivity could not be attributed to genome damage or capsid cleavage. The range in k and the difference in the inactivation mechanism observed in water from the same site could not be explained, but variable constituents in the environmental matrix could have contributed. Thus, a single k may be insufficient for modeling virus inactivation in surface waters.

Head-to-head comparison of the immunogenicity of RotaTeq and Rotarix rotavirus vaccines and factors associated with seroresponse in infants in Bangladesh: a randomised, controlled, open-label, parallel, phase 4 trial.

BACKGROUND: A head-to-head comparison of the most widely used oral rotavirus vaccines has not previously been done, particularly in a high child mortality setting. We therefore aimed to compare the immunogenicity of RotaTeq (Merck, Kenilworth, NJ, USA) and Rotarix (GlaxoSmithKline, Rixensart, Belgium) rotavirus vaccines in the same population and examined risk factors for low seroresponse. METHODS: We did a randomised, controlled, open-label, parallel, phase 4 trial in urban slums within Mirpur and Mohakahli (Dhaka, Bangladesh). We enrolled eligible participants who were healthy infants aged 6 weeks and full-term (ie, >37 weeks' gestation). We randomly assigned participants (1:1), using block randomisation via a computer-generated electronic allocation with block sizes of 8, 16, 24, and 32, to receive either three RotaTeq vaccine doses at ages 6, 10, and 14 weeks or two Rotarix doses at ages 6 and 10 weeks without oral poliovirus vaccine. Coprimary outcomes were the rotavirus-specific IgA seroconversion in both vaccines, and the comparison of the rotavirus IgA seroconversion by salivary secretor phenotype in each vaccine arm. Seroconversion at age 18 weeks in the RotaTeq arm and age of 14 weeks in the Rotarix arm was used to compare the complete series of each vaccine. Seroconversion at age 14 weeks was used to compare two RotaTeq doses versus two Rotarix doses. Seroconversion at age 22 weeks was used to compare the immunogenicity at the same age after receiving the full vaccine series. Safety was assessed for the duration of study participation. This study is registered with ClinicalTrials.gov, NCT02847026. FINDINGS: Between Sept 1 and Dec 8, 2016, a total of 1144 infants were randomly assigned to either the RotaTeq arm (n=571) or Rotarix arm (n=573); 1080 infants (531 in the RotaTeq arm and 549 in the Rotarix arm) completed the study. Rotavirus IgA seroconversion 4 weeks after the full series occurred in 390 (73%) of 531 infants age 18 weeks in the RotaTeq arm and 354 (64%) of 549 infants age 14 weeks in the Rotarix arm (p=0·01). At age 14 weeks, 4 weeks after two doses, RotaTeq recipients had lower seroconversion than Rotarix recipients (268 [50%] of 531 vs 354 [64%] of 549; p<0·0001). However, at age 22 weeks, RotaTeq recipients had higher seroconversion than Rotarix recipients (394 [74%] of 531 vs 278 [51%] of 549; p<0·0001). Among RotaTeq recipients, seroconversion 4 weeks after the third dose was higher than after the second dose (390 [73%] of 531 vs 268 [50%] of 531; p<0·0001]. In the RotaTeq arm, rotavirus IgA seroconversion was lower in non-secretors than in secretors at ages 14 weeks (p=0·08), 18 weeks (p=0·01), and 22 weeks (p=0·02). Similarly, in the Rotarix arm, rotavirus IgA seroconversion was lower in non-secretors than in secretors at ages 14 weeks (p=0·02) and 22 weeks (p=0·01). 65 (11%) of 571 infants had adverse events in the RotaTeq arm compared with 63 (11%) of 573 infants in the Rotarix arm; no adverse events were attributed to the use of either vaccine. One death due to aspiration occurred in the RotaTeq arm, which was not related to the vaccine. INTERPRETATION: RotaTeq induced a higher magnitude and longer duration of rotavirus IgA response than Rotarix in this high child mortality setting. Additional vaccination strategies should be evaluated to overcome the suboptimal performance of current oral rotavirus vaccines in these settings. FUNDING: US Centers for Disease Control and Prevention.

Development and Validation of an Enzyme Immunoassay for Detection and Quantification of SARS-CoV-2 Salivary IgA and IgG.

Oral fluids offer a noninvasive sampling method for the detection of Abs. Quantification of IgA and IgG Abs 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 Abs against the prefusion-stabilized form of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein expressed in suspension-adapted HEK-293 cells. Normalization against total Ab 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 prepandemic 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 endemic 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 Abs were detected earlier in patients with mild COVID-19 symptoms than in severe cases. However, severe cases showed higher salivary Ab titers than those with a mild infection. Salivary IgA titers quickly decreased after 6 wk 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.

Development and validation of an enzyme immunoassay for detection and quantification of SARS-CoV-2 salivary IgA and IgG.

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.

Humoral and Mucosal Immune Responses to Human Norovirus in the Elderly.

BACKGROUND: Most information on mucosal and systemic immune response to norovirus infection is derived from human challenge studies, birth cohort studies, or vaccine trials in healthy adults. However, few data are available on immune responses to norovirus in the elderly. METHODS: To study the mucosal and systemic immune response against norovirus, 43 long-term care facilities were enrolled prospectively in 2010-2014. Baseline saliva samples from 17 facilities, cases and controls up to day 84 from 10 outbreaks, as well as acute and convalescent sera were collected. RESULTS: Norovirus-specific immunoglobulin A (IgA) levels in baseline saliva samples were low and increased in both symptomatic patients and asymptomatic shedders at day 5 after onset during outbreaks. Receiver operating characteristics analysis correctly assigned prior norovirus infection in 23 (92%) of 25 participants. Cases and asymptomatic shedders showed seroconversion for IgG (80%), IgA (78%), and blockade antibodies (87%). Salivary IgA levels strongly correlated with increased convalescent serum IgA titers and blockade antibodies. CONCLUSIONS: Salivary IgA levels strongly correlated with serum IgA titers and blockade antibodies and remained elevated 3 months after a norovirus outbreak. A single salivary sample collected on day 14 could be used to identify recent infection in a suspected outbreak or to monitor population salivary IgA.

Epidemiologic, Virologic, and Host Genetic Factors of Norovirus Outbreaks in Long-term Care Facilities.

BACKGROUND: In the Unites States, long-term care facilities (LTCFs) are the most common setting for norovirus outbreaks. These outbreaks provide a unique opportunity to better characterize the viral and host characteristics of norovirus disease. METHODS: We enrolled 43 LTCFs prospectively to study the epidemiology, virology, and genetic host factors of naturally occurring norovirus outbreaks. Acute and convalescent stool, serum, and saliva samples from cases, exposed and nonexposed controls were collected. Norovirus infection was confirmed using quantitative polymerase chain reaction testing of stool samples or 4-fold increase in serum antibody titers. The presence of histo-blood group antigens (secretor, ABO, and Lewis type) was determined in saliva. RESULTS: Sixty-two cases, 34 exposed controls, and 18 nonexposed controls from 10 norovirus outbreaks were enrolled. Forty-six percent of acute, 27% of convalescent case, and 11% of control stool samples tested norovirus positive. Outbreak genotypes were GII.4 (Den Haag, n = 3; New Orleans, n = 4; and Sydney, n = 2) and GI.1 (n = 1). Viral load in GII.4 Sydney outbreaks was significantly higher than in outbreaks caused by other genotypes; cases and controls shed similar amounts of virus. Forty-seven percent of cases shed virus for ≥ 21 days. Symptomatic infections with GII.4 Den Haag and GII.4 New Orleans were detected among nonsecretor individuals. CONCLUSIONS: Almost half of all symptomatic individuals shed virus for at least 21 days. Viral load was highest in GII.4 viruses that most recently emerged; these viruses also infect the nonsecretor population. These findings will help to guide development of targeted prevention and control measures in the elderly.

Presence of antibodies against genogroup VI norovirus in humans.

BACKGROUND: Noroviruses are important enteric pathogens in humans and animals. Recently, we reported a novel canine norovirus (CaNoV) in dogs with diarrhea belonging to a new genogroup (GVI). No data are available on exposure of humans to this virus. METHODS: Sera from 373 small animal veterinarians and 120 age-matched population controls were tested for IgG antibodies to CaNoV by a recombinant virus like particle based enzyme-linked immunosorbent assay. RESULTS: Antibodies to CaNoV were found in 22.3% of the veterinarians and 5.8% of the control group (p < 0.001). Mean corrected OD450 values for CaNoV antibodies were significantly higher in small animal veterinarians compared to the control group. CONCLUSIONS: These findings suggest that CaNoV may infect humans and small animal veterinarians are at an increased risk for exposure to this virus. Additional studies are needed to assess if this virus is able to cause disease in humans.

Antiviral activity of nucleoside analogues against norovirus.

BACKGROUND: Norovirus (NoV) is the leading cause of epidemic gastroenteritis worldwide. The lack of a cell culture has significantly hampered the development of effective therapies against human NoV. Clinically approved nucleoside and non-nucleoside analogues have been used successfully against RNA viruses. METHODS: In this study, we evaluated the efficacy of four nucleoside analogues (2'-C-MeC, 2'-F-2'-C-MeC, ß-D-N(4)-hydroxycytidine [NHC] and lamivudine) on Norwalk virus (NV) RNA levels and protein expression in NV replicon-harbouring cells (HG23 cells), and their efficacy in blocking murine norovirus (MNV) replication in RAW 264.7 cells. RESULTS: 2'-C-MeC and 2'-F-2'-C-MeC reduced MNV RNA levels and infectivity in RAW 264.7 cells in a concentration- and time-dependent manner. The median effective concentrations (EC(50)) of 2'-C-MeC and 2'-F-2'-C-MeC were 6.9 µM and 12.7 µM, respectively. 2'-C-MeC, 2'-F-2'-C-MeC and NHC reduced NV RNA levels and protein expression in HG23 cells. For the NV replicon, the EC(50) of 2'-C-MeC (1.3 µM) was comparable to the antiviral activity of NHC (1.5 µM) and twofold more potent than 2'-F-2'-C-MeC (3.2 µM). The combination of 2'-C-MeC/ribavirin resulted in modest synergistic activity, whereas NHC/ribavirin was antagonistic for NV replication in HG23 cells. CONCLUSIONS: The antiviral activity of 2'-C-MeC against strains of two different NoV genogroups and the low EC(50) suggest that this nucleoside analogue may be effective against the more prevalent GII NoVs. In the absence of a vaccine, antiviral agents could be an effective intervention to control the spread of human NoV in populations at a high risk for NoV disease.

Effects of different animal waste treatment technologies on detection and viability of porcine enteric viruses.

Enteric pathogens in animal waste that is not properly processed can contaminate the environment and food. The persistence of pathogens in animal waste depends upon the waste treatment technology, but little is known about persistence of porcine viruses. Our objectives were to characterize the porcine enteric viruses (porcine noroviruses [PoNoVs], porcine sapoviruses [PoSaVs], rotavirus A [RV-A], RV-B, and RV-C) in fresh feces or manure and to evaluate the effects of different candidate environmentally superior technologies (ESTs) for animal waste treatment on the detection of these viruses. Untreated manure and samples collected at different stages during and after treatment were obtained from swine farms that used conventional waste management (CWM) and five different candidate ESTs. The RNA from porcine enteric viruses was detected by reverse transcription-PCR and/or seminested PCR; PoSaV and RV-A were also detected by enzyme-linked immunosorbent assay. Cell culture immunofluorescence (CCIF) and experimental inoculation of gnotobiotic (Gn) pigs were used to determine RV-A/C infectivity in posttreatment samples. The PoSaV and RV-A were detected in pretreatment samples from each farm, whereas PoNoV and RV-C were detected in pretreatment feces from three of five and four of five farms using the candidate ESTs, respectively. After treatment, PoSaV RNA was detected only in the samples from the farm using CWM and not from the farms using the candidate ESTs. RV-A and RV-C RNAs were detected in four of five and three of four candidate ESTs, respectively, after treatment, but infectious particles were not detected by CCIF, nor were clinical signs or seroconversion detected in inoculated Gn pigs. These results indicate that only RV-A/C RNA, but no viral infectivity, was detected after treatment. Our findings address a public health concern regarding environmental quality surrounding swine production units.