Norovirus: Facts and Reflections from Past, Present, and Future.

Human Norovirus is currently the main viral cause of acute gastroenteritis (AGEs) in most countries worldwide. Nearly 50 years after the discovery of the "Norwalk virus" by Kapikian and colleagues, the scientific and medical community continue to generate new knowledge on the full biological and disease spectrum of Norovirus infection. Nevertheless, several areas remain incompletely understood due to the serious constraints to effectively replicate and propagate the virus. Here, we present a narrated historic perspective and summarize our current knowledge, including insights and reflections on current points of interest for a broad medical community, including clinical and molecular epidemiology, viral-host-microbiota interactions, antivirals, and vaccine prototypes. We also include a reflection on the present and future impacts of the COVID-19 pandemic on Norovirus infection and disease.

Analysis by rotavirus gene 6 reverse transcriptase-polymerase chain reaction assay of rotavirus-positive gastroenteritis cases observed during the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST).

During the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST), the period between the administration of dose 1 through 13 days after the administration of dose 3, there were more wild-type rotavirus gastroenteritis (RVGE) cases among vaccine recipients compared with placebo recipients using the protocol-specified microbiological plaque assay in the clinical-efficacy cohort, a subset of subjects where vaccine efficacy against RVGE of any severity was assessed. In this study, a rotavirus genome segment 6-based reverse transcriptase-polymerase chain reaction assay was applied post hoc to clarify the accuracy of type categorization of all these RVGE cases in vaccine recipients during the vaccination phase of REST. The assay characterized 147 (90%) of 163 re-assayed RVGE cases or rotavirus-associated health care contacts as type-determinable: either wild-type or vaccine-type rotavirus strains. In the clinical-efficacy cohort (N = 5673), 19 (18.8%) of 101 samples from RVGE cases contained wild-type rotavirus, 70 (69.3%) vaccine virus, and 12 (11.9%) were indeterminable. In the large-scale cohort (N = 68,038), 10 (34.5%) of 29 samples from RVGE-related health care contacts contained wild-type rotavirus strains, 15 (51.7%) vaccine-type rotavirus strains, and 4 (13.8%) were indeterminable. Of the 33 samples from RVGE cases in placebo recipients, all were confirmed to contain wild-type rotaviruses. Altogether, this post-hoc re-evaluation showed that the majority (75%) of type-determinable RVGE cases or health care contacts that occurred during the vaccination phase of REST in vaccine recipients were associated with vaccine-type rotavirus strains rather than wild-type rotavirus strains.

The public health leadership certificate: a public health and primary care interprofessional training opportunity.

This article describes a public health leadership certificate curriculum developed by the Commonwealth Public Health Training Center for employees in public health and medical trainees in primary care to share didactic and experiential learning. As part of the program, trainees are involved in improving the health of their communities and thus gain a blended perspective on the effectiveness of interprofessional teams in improving population health. The certificate curriculum includes eight one-credit-hour didactic courses offered through an MPH program and a two-credit-hour, community-based participatory research project conducted by teams of trainees under the mentorship of health district directors. Fiscal sustainability is achieved by sharing didactic courses with MPH degree students, thereby enabling trainees to take advantage of a reduced, continuing education tuition rate. Public health employee and primary care trainees jointly learn knowledge and skills required for community health improvement in interprofessional teams and gain an integrated perspective through opportunities to question assumptions and broaden disciplinary approaches. At the same time, the required community projects have benefited public health in Virginia.

Burden of rotavirus hospitalisations in young children in three paediatric hospitals in the United States determined by active surveillance compared to standard indirect methods.

AIM: The number of rotavirus hospitalisations is usually estimated from assigned diagnosis codes for gastroenteritis despite lack of validation for these indirect methods. Reliable estimates before and after introduction of vaccines are needed to quantify the absolute impact of new immunisation programs. METHODS: This 2-year study conducted at three hospitals prior to the licensure of the rotavirus vaccines in the USA compared two indirect methods for estimating hospitalisations for rotavirus gastroenteritis with estimates derived from prospective recruitment of children presenting with diarrhoea, vomiting or fever. For active surveillance, rotavirus gastroenteritis was confirmed by demonstration of stool antigen. The indirect residual and proportional methods assumed rotavirus to have caused a proportion of hospitalisations coded as acute gastroenteritis identified from computerised records. RESULTS: There were 447 rotavirus hospitalisations among inpatients 31 days through 4 years of age admitted with vomiting and/or diarrhoea, compared with 306 and 228 hospitalisations identified by the two indirect methods. Only 52% of children hospitalised with gastroenteritis received a qualifying diagnosis code at discharge. Relative to active surveillance, the sensitivity and specificity (95% confidence interval (CI)) in identifying rotavirus-attributable hospitalisations was 45% (95% CI: 43-48%) and 89% (88-90%) for the residual method and 34% (30-39%) and 92% (90-94%) for the proportional method. CONCLUSIONS: Many children admitted to the hospital with diarrhoea, vomiting or fever were not assigned discharge codes for acute gastroenteritis. Consequently, standard indirect methods missed a substantial number of rotavirus-associated hospitalisations, thereby underestimating the absolute number of children who could potentially benefit from vaccination.

Efficacy of the pentavalent rotavirus vaccine, RotaTeq® (RV5), between doses of a 3-dose series and with less than 3 doses (incomplete regimen).

Post-hoc analyses of the Rotavirus Efficacy and Safety Trial (REST) were conducted to determine whether the pentavalent rotavirus vaccine (RV5) confers early protection against rotavirus gastroenteritis (RVGE) before completion of the 3-dose regimen. To evaluate the efficacy of RV5 between doses in reducing the rates of RVGE-related hospitalizations and emergency department (ED) visits in infants who ultimately received all 3 doses of RV5/placebo, events occurring from 2 weeks after the first and second doses to receipt of the subsequent dose (Analysis A) and events occurring from 2 weeks after the first and second doses to 2 weeks after the subsequent dose (Analysis B) were analyzed. In Analysis A, RV5 reduced the rates of combined hospitalizations and ED visits for G1-G4 RVGE or RVGE regardless of serotype between doses 1 and 2 by 100% (95% confidence interval [CI]: 72-100%) or 82% (95% CI: 39-97%), respectively, and between doses 2 and 3, RV5 reduced the rates of combined hospitalizations and ED visits for G1-G4 RVGE or RVGE regardless of serotype by 91% (95% CI: 63-99%) or 84% (95% CI: 54-96%), respectively. Similar rate reductions were observed in Analysis B. These data suggest that RV5 provides a high level of protection between doses against hospitalizations and ED visits for RVGE starting as early as 14 days after the first dose.

Rotavirus genotypes among hospitalized children in Egypt, 2000-2002.

Rotavirus type surveillance is essential to assess the success of rotavirus vaccines. Rotavirus strains collected in 2000-2002 during hospital-based surveillance for diarrhea in Egyptian children were genotyped. Of the 259 (25.2%) rotavirus-positive specimens, 82.4% were common strains (G1p[8], G2p[4], G4p[8]), and the emergent G9 type was detected in 5.3% of samples.

Robustness of the healthcare utilization results from the Rotavirus Efficacy and Safety Trial (REST) evaluating the human-bovine (WC3) reassortant pentavalent rotavirus vaccine (RV5).

BACKGROUND: The Rotavirus Efficacy and Safety Trial was a placebo-controlled Phase III study that evaluated the safety and efficacy of a three-dose pentavalent rotavirus vaccine (RV5) including its effect on healthcare utilization for rotavirus gastroenteritis (RVGE). The per-protocol (PP) analyses, which counted events occurring 14 days after dose 3 among infants without protocol violations, have already been published. This paper evaluates the consistency of the healthcare utilization results based on the modified intention to treat (MITT) analyses with the PP analyses. The MITT analyses include all infants receiving at least one dose of vaccine or placebo and follow-up begins after dose 1. The paper also explores the consistency of the results for different subgroups of the study population with different types of surveillance. METHODS: Data on healthcare utilization for acute gastroenteritis were collected via telephone interviews after administration of the first dose. Parents were either contacted every 6 weeks or every 2 weeks depending on the substudy in which they were enrolled. Those contacted every 2 weeks were also asked to complete symptom diaries. Poisson regression was used to evaluate the effect of RV5 on the rates of RVGE-associated healthcare encounters in all of the analyses. RESULTS: In the first 2 years after vaccination, RV5 reduced the combined rate of hospitalizations and emergency department (ED) visits 88.9% (95% CI: 84.9, 91.9) for all RVGE regardless of serotype in the MITT analysis compared with a 94.5% (95% CI: 91.2, 96.6) reduction based on the G1-G4 PP analysis. By type of surveillance, the rate reductions for the G1-G4 PP analysis were 91.0% (95% CI: 81.7, 95.5) and 95.9% (95% CI: 92.2, 97.8) among parents contacted every 2 weeks (number evaluable = 4,451) and every 6 weeks (number evaluable = 52,683) respectively. CONCLUSIONS: Our analyses demonstrated that the effect of RV5 on reducing the rate of hospitalizations and ED visits based on the MITT analyses were generally consistent with the PP analyses. The rate of events for subgroups with different intensities of surveillance differed but the effect of RV5 on the relative rate reductions were consistent with the results that have already been published. TRIAL REGISTRATION: ClinicalTrials.gov number, NCT00090233.

A cross-sectional evaluation of the first cohort of young adults conceived by in vitro fertilization in the United States.

OBJECTIVE: To assess the quality of life and susceptibility for chronic disease development of the oldest generation of young adults conceived by IVF in the U.S. DESIGN: Cross-sectional. SETTING: Single tertiary clinic. PATIENT(S): Young adults conceived by standard IVF between 1981 and 1990. INTERVENTION(S): Self-administered questionnaire. MAIN OUTCOME MEASURE(S): Indicators of physical, psychologic, and behavioral health. RESULT(S): A total of 173 (31%) of 560 eligible young adults completed the questionnaire. Mean age was 21.2 years (range 18-26 years) and male-to-female ratio was 3:4. A limited number were conceived through gamete donation but none through oocyte/embryo micromanipulation. Prevalence rates of overweight and obesity were 35% and 10%, respectively. More than 65% were ever diagnosed with a chronic condition; most diagnoses were psychiatric, ocular, respiratory, and cardiometabolic in nature. Almost 40% of respondents were lifetime smokers, 62% reported binge drinking in the previous year, and >90% were physically active in the preceding month. Survey participants were mostly similar to a subsample of the 1999-2004 National Health and Nutrition Examination Survey on selected health indicators. CONCLUSION(S): Young adults conceived by IVF appear to be healthy and well adjusted, although the preponderance of psychologic health problems requires further investigation.

Impact of polycystic ovary syndrome on selected indicators of in vitro fertilization and intracytoplasmic sperm injection treatment success.

OBJECTIVES: To examine the effect of polycystic ovary syndrome (PCOS), a marker of the metabolic syndrome, on selected indicators of in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatment success. METHODS: A retrospective cohort study was conducted using existing data on 69 IVF/ICSI treatment cycles undergone by PCOS women and a caliper-matched sample of 69 IVF/ICSI treatment cycles undergone by non-PCOS women over a 7-year period at a major fertility treatment center. Matching criteria were age and date at IVF/ICSI treatment initiation. Process and outcome measures were used to define successful IVF/ICSI treatment. Statistical significance was determined at an alpha level of 0.05. RESULTS: The total number of oocytes and the number of immature oocytes retrieved in the process of an IVF/ICSI cycle were significantly higher in the context of PCOS. No significant differences were observed among PCOS and non-PCOS groups on various IVF/ICSI cycle outcomes, including high-grade embryo, pregnancy achievement, miscarriage, and live birth status. CONCLUSIONS: Although IVF/ICSI yields more oocytes in the context of PCOS, IVF/ICSI outcomes do not differ significantly by PCOS status. Prospective cohort studies are needed to examine short-term and long-term health effects of PCOS in the context of IVF/ICSI.

Automated assessment of ovarian follicles using a novel three-dimensional ultrasound software.

OBJECTIVE: To evaluate the accuracy, efficiency, and applicability of a new automated method of follicular assessment. DESIGN: Retrospective review of three-dimensional ultrasonographic ovarian volumes. SETTING: Academic center. PATIENT(S): Three-dimensional ovarian volumes from patients undergoing IVF. INTERVENTION(S): Three-dimensional volumes of gonadotropin-stimulated ovaries and simulated ovarian follicles (SOFs) of known volume were evaluated with the new automated follicular assessment software (SonoAVC). MAIN OUTCOME MEASURE(S): [1] Maximum absolute error for the SonoAVC in assessing the volumes of the SOFs; [2] correlations between the automated and manual follicular measurements; [3] time required to analyze all of the follicles in a given ovarian volume. RESULT(S): The SOF of 4, 6, and 10 mm were evaluated. The SonoAVC's maximum absolute error for the volumes of the 4, 6, and 10 mm SOFs was 0 (0%), 0.01 (8.3%), and 0.02 (3.8%) cc, respectively. Three hundred forty-seven follicles, ranging in diameter from 2.3-32 mm, were evaluated. The correlation coefficient for the SonoAVC-generated relaxed follicular diameter and the manual measured mean follicular diameter was 0.99. The time required to analyze all of the follicles in a given ovarian volume for the automated and manual method was 133 versus 361 seconds, respectively. CONCLUSION(S): The SonoAVC proved to be a very accurate and efficient way to measure ovarian follicles. The measurements obtained by the SonoAVC correlated extremely well with the manual measurements we obtained.

Burden of rotavirus disease among children visiting pediatric emergency departments in Cincinnati, Ohio, and Oakland, California, in 1999-2000.

OBJECTIVE: We assessed the incidence of rotavirus disease requiring an emergency department visit among children <5 years of age. METHODS: We conducted active surveillance for acute gastroenteritis in pediatric emergency departments in Cincinnati, Ohio, and Oakland, California, from March 1999 to May 2000, among children 2 weeks to 59 months of age with acute diarrhea and/or vomiting. We obtained clinical and demographic information from participants and tested their stool specimens for rotavirus. RESULTS: Approximately 9% of all emergency department visits at the study sites were attributable to acute gastroenteritis. A total of 1433 children were eligible at the 2 sites; 85% were enrolled and 68% provided a stool specimen. Overall, rotavirus was detected in specimens from 27% of children (30% in Cincinnati and 24% in Oakland). Rotavirus detection was higher in bulk stools, compared with rectal swabs, at both Cincinnati (37% vs 23%) and Oakland (46% vs 18%). Patients with rotavirus had more-severe disease than did those with nonrotavirus gastroenteritis. We estimated that the mean annual incidence of emergency department visits attributable to rotavirus was 12 cases per 1000 children in Cincinnati and 15 cases per 1000 children in Oakland. Through extrapolation, we estimated that rotavirus infection causes approximately 260,910 emergency department visits per year among US children. CONCLUSION: Active surveillance demonstrated that the burden of laboratory-confirmed rotavirus disease treated in emergency department settings among US children is substantial and greater than estimated previously.

Inhibition of norovirus replication by morpholino oligomers targeting the 5'-end of the genome.

Noroviruses are an important cause of non-bacterial epidemic gastroenteritis, but no specific antiviral therapies are available. We investigated the inhibitory effect of phosphorodiamidiate morpholino oligomers (PMOs) targeted against norovirus sequences. A panel of peptide-conjugated PMOs (PPMOs) specific for the murine norovirus (MNV) genome was developed, and two PPMO compounds directed against the first AUG of the ORF1 coding sequence near the 5'-end of the genome proved effective in inhibiting MNV replication in cells. A consensus PPMO (designated Noro 1.1), designed to target the corresponding region of several diverse human norovirus genotypes, decreased the efficiency of protein translation in a cell-free luciferase reporter assay and inhibited Norwalk virus protein expression in replicon-bearing cells. Our data suggest that PPMOs directed against the relatively conserved 5'-end of the norovirus genome may show broad antiviral activity against this genetically diverse group of viruses.

Virus-specific antiviral treatment for controlling severe and fatal outbreaks of feline calicivirus infection.

OBJECTIVE: To test the life-sparing and therapeutic effect of a parenterally administered virus-specific antiviral phosphorodiamidate morpholino oligomer (PMO) for treating kittens during outbreaks of severe viral disease. ANIMALS: 112 kittens of various sex and age in 4 trials involving 3 outbreaks of naturally developing caliciviral disease. PROCEDURES: Each trial provided an opportunity to investigate the disease. A calicivirus isolated from the liver of a cat that died with hemorrhage and hepatitis was sequenced, and a PMO that had sequence specificity complementary to a 5' region was synthesized. In vitro efficacy of the PMO was tested against the isolate, followed by 3 trials in outbreaks of severe caliciviral disease. The PMO was administered starting on day 1 of disease onset (0.7 to 5.0 mg/kg, SC, q 24 h) and continuing for up to 7 days. Survival time, clinical recovery, and caliciviral shedding were compared by use of various antiviral dosages. In a fourth trial involving nonfatal disease, a control treatment was administered for comparison. RESULTS: In vitro blockage of caliciviral replication by the PMO was dose dependent. In trials 1 to 3 in which survival was the endpoint, 47 of 59 cats receiving PMO survived but only 3 of 31 survived without PMO treatment. Antiviral treatment reduced viral shedding and hastened clinical recovery, as measured by weight gains and clinical condition. CONCLUSIONS AND CLINICAL RELEVANCE: These data provided evidence that virus-specific PMOs were effective in treating kittens with severe Vesivirus disease and suggested a broader application for other viruses and species, including humans.

Rotavirus antigenemia in children is associated with viremia.

BACKGROUND: Antigenemia is commonly detected in rotavirus-infected children. Although rotavirus RNA has been detected in serum, definitive proof of rotavirus viremia has not been shown. We aimed to analyze a defined patient population to determine if infectious virus could be detected in sera from children with rotavirus antigenemia. METHODS AND FINDINGS: Serum samples obtained upon hospitalization from children with gastroenteritis (57 stool rotavirus-positive and 41 rotavirus-negative), children with diagnosed bronchiolitis of known (n = 58) or unknown (n = 17) viral etiology, children with noninfectious, nonchronic conditions (n = 17), and healthy adults (n = 28) were tested for rotavirus antigen by enzyme immunoassay (EIA). Results of serum antigen testing were assessed for association with clinical and immunological attributes of the children. Rotavirus antigenemia was detected in 90% (51/57) of children with rotavirus-positive stools, in 89% (8/9) of children without diarrhea but with rotavirus-positive stools, in 12% (2/17) of children with bronchiolitis of unknown etiology without gastroenteritis, and in 12% (5/41) of children with gastroenteritis but with rotavirus-negative stools. Antigenemia was not detected in sera from children with noninfectious nonchronic conditions, children with bronchiolitis of known etiology and no gastroenteritis, or healthy adults. Neither age nor timing of serum collection within eight days after onset of gastroenteritis significantly affected levels of antigenemia, and there was no correlation between antigenemia and viral genotype. However, there was a negative correlation between serum rotavirus antigen and acute rotavirus-specific serum IgA (r = -0.44, p = 0.025) and IgG (r = -0.40, p = 0.01) titers. We examined 11 antigen-positive and nine antigen-negative sera for infectious virus after three blind serial passages in HT-29 cells using immunofluorescence staining for rotavirus structural and nonstructural proteins. Infectious virus was detected in 11/11 (100%) sera from serum antigen-positive children and in two out of nine (22%) sera samples from antigen-negative children (p = 0.002). CONCLUSIONS: Most children infected with rotavirus are viremic. The presence of viremia is directly related to the detection of antigenemia and is independent of the presence of diarrhea. Antigenemia load is inversely related to the titer of antirotavirus antibody in the serum. The finding of infectious rotavirus in the blood suggests extraintestinal involvement in rotavirus pathogenesis; however, the impact of rotavirus viremia on clinical manifestations of infection is unknown.

Inhibition of replication and transcription activator and latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus by morpholino oligomers.

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma and primary effusion lymphoma (PEL). The KSHV replication and transcription activator (RTA) and latency-associated nuclear antigen (LANA) play key roles in activating KSHV lytic replication and maintaining KSHV latency, respectively. Phosphorodiamidate morpholino oligomers (PMO) are similar to short single-stranded DNA oligomers, but possess a modified backbone that confers highly specific binding and resistance to nucleases. In this study, RTA and LANA mRNA in PEL cells were targeted by antisense peptide-conjugated PMO (P-PMO) in an effort to suppress KSHV replication. Highly efficient P-PMO uptake by PEL cells was observed. Treatment of PEL cells with a RTA P-PMO (RP1) reduced RTA expression in a dose-dependent and sequence-specific manner, and also caused a significant decrease in several KSHV early and late gene products, including vIL-6, vIRF-1, and ORF-K8.1A. KSHV viral DNA levels were reduced both in cells and culture supernatants of RP1 P-PMO-treated cells, indicating that KSHV lytic replication was suppressed. Treatment of BCBL-1 cells with P-PMO against LANA resulted in a reduction of LANA expression. Cell viability assays detected no cytotoxicity from P-PMO alone, within the concentration range used for the experiments in this study. These results suggest that RP1 P-PMO can specifically block KSHV replication, and further study is warranted.

Efficacy of a pentavalent rotavirus vaccine in reducing rotavirus-associated health care utilization across three regions (11 countries).

OBJECTIVE: To evaluate the effect of a human-bovine reassortant pentavalent rotavirus vaccine (PRV) on health care encounters in nearly 70 000 subjects randomized in three regions - Europe, the United States, and Latin America/the Caribbean - in the Rotavirus Efficacy and Safety Trial (REST). METHODS: Healthy 6- to 12-week-old infants received 3 doses of PRV or placebo at 4- to 10-week intervals. The exact binomial method for ratios of Poisson counts was used to evaluate the effect of PRV on the rate of rotavirus-related hospitalizations and emergency department (ED) visits involving rotavirus G-types 1-4 occurring > or =14 days after the third dose of vaccine for up to 2 years. RESULTS: In fully vaccinated infants, reductions in rotavirus-associated hospitalizations and ED visits were 94.7% (95% CI: 90.9, 96.9) in Europe, 94.9% (95% CI: 84.0, 98.9) in the United States, and 90.0% (95% CI: 29.4, 99.8) in the Latin American/Caribbean regions. CONCLUSIONS: PRV reduced hospitalizations and ED visits within each region in REST. Results were consistent across regions and across the overall study cohort.

The pentavalent rotavirus vaccine, RotaTeq.

Vaccination against rotavirus disease has been a global preventive health priority since rotaviruses were first discovered in the 1970s. The first licensed rotavirus vaccine, RRV-TV, was removed from the market shortly after licensure because of an unexpected serious adverse event, intussusception. A pentavalent rotavirus vaccine, PRV, is the second FDA-licensed rotavirus vaccine and does not signal the risk observed with RRV-TV. The properties and large safety study results of PRV are reviewed here.