A Decade of Chronic Norovirus Infection Surveillance at the NIH Clinical Research Center: Clinical Characteristics, Molecular Epidemiology, and Replication.

BACKGROUND: Noroviruses are an important viral cause of chronic diarrhea in immunocompromised individuals. METHOD: We collected norovirus-positive stool samples (n=448) from immunocompromised patients (n=88) at the National Institutes of Health Clinical Research Center, U.S. from 2010-2022. We assessed clinical characteristics of the cohort, norovirus molecular epidemiology, and infectivity of norovirus specimens in human intestinal enteroids (HIEs) monolayers. RESULTS: Thirty-nine of the 88 patients had sequential stool samples that allowed documentation of chronic norovirus infection with shedding levels ranging from 104 to 1011 genome copies/g of stool. The majority with confirmed chronic norovirus infection in this cohort (32/39, 82%) had clinical evidence of an inborn error of immunity (13 identified monogenic diseases), most with combined immunodeficiency (15 of 32) or common variable immunodeficiency (11 of 32). Noroviruses detected in the cohort were genetically diverse: both Genogroup I (GI.2, GI.3, GI.5, and GI.6) and Genogroup II (GII.1-GII.4, GII.6, GII.7, GII.12, GII.14, and GII.17) genotypes were detected, with GII.4 variants (Osaka, Apeldoorn, Den Haag, New Orleans, and Sydney) predominant (51 of 88, 57.9%). Viruses belonging to the GII.4 Sydney variant group that replicated in HIEs (n=9) showed a higher fold-increase in RNA genome copies during infection compared to others that replicated. CONCLUSIONS: Genetically and biologically diverse noroviruses established chronic infection in individuals with both inborn and acquired immunologic defects enrolled in an NIH surveillance study spanning 12 years, demonstrating the unique nature of each virus and host interaction.

A narrative review of norovirus epidemiology, biology, and challenges to vaccine development.

Norovirus is a leading cause of acute gastroenteritis (AGE) globally. AGE resulting from norovirus causes significant morbidity and mortality in countries of all income levels, particularly among young children and older adults. Prevention of norovirus AGE represents a unique challenge as the virus is genetically diverse with multiple genogroups and genotypes cocirculating globally and causing disease in humans. Variants of the GII.4 genotype are typically the most common genotype, and other genotypes cause varying amounts of disease year-to-year, with GII.2, GII.3, and GII.6 most prevalent in recent years. Noroviruses are primarily transmitted via the fecal-oral route and only a very small number of virions are required for infection, which makes outbreaks of norovirus extremely difficult to control when they occur. Settings like long-term care facilities, daycares, and hospitals are at high risk of outbreaks and can have very high attack rates resulting in substantial costs and disease burden. Severe cases of norovirus AGE are most common in vulnerable patient populations, such as infants, the elderly, and immunocompromised individuals, with available treatments limited to rehydration therapies and supportive care. To date, there are no FDA-approved norovirus vaccines; however, several candidates are currently in development. Given the substantial human and economic burden associated with norovirus AGE, a vaccine to prevent morbidity and mortality and protect vulnerable populations could have a significant impact on global public health.

Norovirus evolves as one or more distinct clonal populations in immunocompromised hosts.

Noroviruses are a major cause of acute gastroenteritis worldwide and can establish chronic infection in immunocompromised individuals. To investigate the mechanisms of norovirus evolution during chronic infection, we selected seven representative patients from a National Institutes of Health study cohort who sustained norovirus infection for periods ranging from 73 to 1,492 days. Six patients shed viruses belonging to a single genotype (GII.2[PNA], GII.4 New Orleans[P4], GII.4 Den Haag[P4], GII.3[P21], GII.6[P7], or GII.14[P7]) over the period examined, while one patient sequentially shed two genotypes (GII.6[P7] followed by GII.4 Sydney[P31]). Norovirus genomes from consecutive stool samples were sequenced at high resolution (>3,300 reads/nucleotide position) using the Illumina platform and subjected to bioinformatics analysis. Norovirus sequences could be resolved into one or more discrete clonal RNA genomes that persisted within these patients over time. Phylogenetic analyses inferred that clonal populations originated from a single founder virus and not by reinfection with community strains. Estimated evolutionary rates of clonal populations during persistent infection were similar to those of noroviruses from acute infection in the global database, suggesting that inherently higher RNA-dependent polymerase error rates were not associated with the ability to persist. The high-resolution analysis of norovirus diversity and evolution at the population level described here should allow a better understanding of adaptive mutations sustained during chronic infection. IMPORTANCE Noroviruses are an important cause of chronic diarrhea in patients with compromised immune systems. Presently, there are no effective therapies to clear the virus, which can persist for years in the intestinal tract. The goal of our study was to develop a better understanding of the norovirus strains that are associated with these long-term infections. With the remarkable diversity of norovirus strains detected in the immunocompromised patient cohort we studied, it appears that most, if not all, noroviruses circulating in nature may have the capacity to establish a chronic infection when a person is unable to mount an effective immune response. Our work is the most comprehensive genetic data set generated to date in which near full-length genomes from noroviruses associated with chronic infection were analyzed by high-resolution next-generation sequencing. Analysis of this data set led to our discovery that certain patients in our cohort were shedding noroviruses that could be subdivided into distinct haplotypes or populations of viruses that were co-evolving independently. The ability to track haplotypes of noroviruses during chronic infection will allow us to fine-tune our understanding of how the virus adapts and maintains itself in the human host, and how selective pressures such as antiviral drugs can affect these distinct populations.

Association of Secretor Status and Recent Norovirus Infection With Gut Microbiome Diversity Metrics in a Veterans Affairs Population.

Norovirus infection causing acute gastroenteritis could lead to adverse effects on the gut microbiome. We assessed the association of microbiome diversity with norovirus infection and secretor status in patients from Veterans Affairs medical centers. Alpha diversity metrics were lower among patients with acute gastroenteritis but were similar for other comparisons.

Modeling an integrated HIV prevention and care continuum to achieve the Ending the HIV Epidemic goals.

OBJECTIVE: We sought to evaluate, which combinations of HIV prevention and care activities would have the greatest impact towards reaching the US Ending the HIV Epidemic (EHE) plan goals of HIV incidence reduction. DESIGN: A stochastic network-based HIV transmission model for men who have sex with men (MSM), calibrated to surveillance estimates in the Atlanta area, a focal EHE jurisdiction. METHODS: Model scenarios varied HIV screening rates under different assumptions of how HIV-negative MSM would be linked to PrEP initiation, and rates of HIV care linkage and retention for those screening positive. RESULTS: A ten-fold relative increase in HIV screening rates (to approximately biannual screening for black and Hispanic MSM and quarterly for white MSM) would lead to 43% of infections averted if integrated with PrEP initiation. Improvements focused only on black MSM would achieve nearly the same outcome (37% of infections averted). Improvements to HIV care retention would avert 41% of infections if retention rates were improved ten-fold. If both screening and retention were jointly improved ten-fold, up to 74% of cumulative infections would be averted. Under this scenario, it would take 4 years to meet the 75% EHE goal and 12 years to meet the 90% goal for Atlanta MSM. CONCLUSION: Reaching the EHE 75% incidence reduction goals by their target dates will require immediate and substantial improvements in HIV screening, PrEP, and ART care retention. Meeting these EHE goals in target jurisdictions like Atlanta will be possible only by addressing the HIV service needs of black MSM.

Use of barley or corn silage when fed with barley, corn, or a blend of barley and corn on growth performance, nutrient utilization, and carcass characteristics of finishing beef cattle.

The objective of this study was to evaluate the effects of the source of silage, cereal grain, and their interaction on growth performance, digestibility, and carcass characteristics of finishing beef cattle. Using a completely randomized design within an 89-d finishing study, 288 steers were randomly assigned to 1 of 24 pens (12 steers/pen) with average steer body weight (BW) within a pen of 464 kg ± 1.7 kg (mean ± SD). Diets were arranged in a 2 × 3 factorial with corn silage (CS) or barley silage (BS) included at 8% (dry matter [DM] basis). Within each silage source, diets contained dry-rolled barley grain (BG; 86% of DM), dry-rolled corn grain (CG; 85% of DM), or an equal blend of BG and CG (BCG; 85% of DM). Total tract digestibility of nutrients was estimated from fecal samples using near-infrared spectroscopy. Data were analyzed with pen as the experimental unit using the Mixed Model of SAS with the fixed effects of silage, grain, and the two-way interaction. Carcass and fecal kernel data were analyzed using GLIMMIX utilizing the same model. There were no interactions detected between silage and grain source. Feeding CG increased (P < 0.01) DM intake by 0.8 and 0.6 kg/d relative to BG and BCG, respectively. Gain-to-feed ratio was greater (P = 0.04) for BG (0.172 kg/kg) than CG (0.162 kg/kg) but did not differ from BCG (0.165 kg/kg). Furthermore, average daily gain (2.07 kg/d) and final body weight did not differ among treatments (P ≥ 0.25). Hot carcass weight (HCW) was 6.2 kg greater (372.2 vs. 366.0 kg; P < 0.01) and dressing percentage was 0.57 percentage units greater (59.53 vs. 58.96 %; P = 0.04) for steers fed CS than BS, respectively. There was no effect of dietary treatment on the severity of liver abscesses (P ≥ 0.20) with 72.0% of carcasses having clear livers, 24.4% with minor liver abscesses, and 3.6% with severe liver abscesses. Digestibility of DM, organic matter, crude protein, neutral detergent fiber, and starch were greater for BG (P < 0.01) than CG or BCG. As expected, grain source affected the appearance of grain kernels in the feces (P ≤ 0.04). Feeding CS silage increased the appearance of fractured corn kernels (P = 0.04), while feeding BS increased fiber appearance in the feces (P = 0.02). Current results indicate that when dry rolled, feeding BG resulted in improved performance and digestibility compared with CG and BCG. Even at low inclusion levels (8% of DM), CS resulted in improved carcass characteristics relative to BS.

Human norovirus targets enteroendocrine epithelial cells in the small intestine.

Human noroviruses are a major cause of diarrheal illness, but pathogenesis is poorly understood. Here, we investigate the cellular tropism of norovirus in specimens from four immunocompromised patients. Abundant norovirus antigen and RNA are detected throughout the small intestinal tract in jejunal and ileal tissue from one pediatric intestinal transplant recipient with severe gastroenteritis. Negative-sense viral RNA, a marker of active viral replication, is found predominantly in intestinal epithelial cells, with chromogranin A-positive enteroendocrine cells (EECs) identified as a permissive cell type in this patient. These findings are consistent with the detection of norovirus-positive EECs in the other three immunocompromised patients. Investigation of the signaling pathways induced in EECs that mediate communication between the gut and brain may clarify mechanisms of pathogenesis and lead to the development of in vitro model systems in which to evaluate norovirus vaccines and treatment.

Effect of feeding barley or corn silage with dry-rolled barley, corn, or a blend of barley and corn grain on rumen fermentation, total tract digestibility, and nitrogen balance for finishing beef heifers.

Five ruminally cannulated heifers were used in an incomplete 6 × 6 Latin square design to determine the effects of cereal silage (barley vs. corn), cereal grain (barley vs. corn vs. a 50:50 blend of barley and corn), and their interaction (S × G) on dry matter intake, ruminal fermentation, total tract digestibility, nitrogen balance, and in situ degradation. Corn silage (CS) or barley silage (BS) was included at 8% of dietary dry matter (DM). Within each silage source, diets contained (DM basis) either dry-rolled barley (BG; 86%), dry-rolled corn (CG; 85%), or an equal blend of barley and corn (BLEND; 85%). Periods were 25 d, with 5 d of dietary transition, 13 d of dietary adaptation, and 7 d of data and sample collection. Samples collected included feed and refusals, total urine and feces, and ruminal fluid. All data were analyzed using the Mixed model of SAS with the fixed effects of silage, grain, and the S × G. Dry matter intake (P ≥ 0.19) and mean ruminal pH (P ≥ 0.096) were not affected by the silage, grain, or S × G. Total short-chain fatty acid concentrations were greater for BLEND than BG or CG (grain, P = 0.003) and for CS (silage, P = 0.009) relative to BS. The molar proportion of acetate was greater for BS-BG and BS-CG (S × G, P < 0.001), while molar proportion of propionate was greater for CS-BG (S × G, P < 0.001) relative to other silage and grain source combinations. Rumen ammonia-N concentration was greater for CG than BG, or BLEND (grain, P < 0.001), and greater for CS compared to BS (silage, P = 0.023). Apparent total tract digestibility of DM, organic matter, neutral detergent fiber, starch, and gross energy were greatest for BG (grain, P ≤ 0.035). Digestible energy content (Mcal/kg) was greater for BG (grain, P = 0.029) than CG and BLEND. Total nitrogen retention (g/d and % of intake) was greatest for CS-BG (S × G, P ≤ 0.033) relative to all other treatments. In situ degradation rates of DM, crude protein, and starch were greater for BG than CG (P ≤ 0.004). The potentially degradable fraction of DM, crude protein, and starch was greater for CG (P ≤ 0.031), while the undegradable fraction was greater for BG (P ≤ 0.046). For silage sources, CS had greater 24 h in situ DM digestibility (P = 0.009) and starch digestibility (24, 48, and 72 h incubations, P ≤ 0.034) relative to BS. Results suggest that while feeding a combination of CS and BG promotes propionate production and greater N retention; few other additive effects were observed.

Comparative Transcriptomic Response of Primary and Immortalized Macrophages to Murine Norovirus Infection.

Murine norovirus (NoV) is genetically similar to human NoV and offers both an efficient in vitro cell culture system and an animal model by which to investigate the molecular basis of replication. In this study, we present a detailed global view of host alterations to cellular pathways that occur during the progression of a NoV infection. This was accomplished for both Mus musculus BALB/c-derived RAW264.7 (RAW) cells, an immortalized cell line widely used in in vitro replication studies, and primary bone marrow-derived macrophages (BMDM), representing a permissive in vivo target cell in the host. Murine NoV replicated in both cell types, although detected genome copies were approximately one log lower in BMDM compared with RAW cells. RAW and BMDM cells shared an IRF3/7-based IFN response that occurred early in infection. In RAW cells, transcriptional upregulation and INF-ß expression were not coupled in that a significant delay in the detection of secreted INF-ß was observed. In contrast, primary BMDM showed an early upregulation of transcripts and immediate release of INF-ß that might account for lower virus yield. Differences in the transcriptional pathway responses included a marked decrease in expression of key genes in the cell cycle and lipid pathways in RAW cells compared with that of BMDM. Our comparative analysis indicates the existence of varying host responses to virus infection in populations of permissive cells. Awareness of these differences at the gene level will be important in the application of a given permissive culture system to the study of NoV immunity, pathogenesis, and drug development.

Static and Evolving Norovirus Genotypes: Implications for Epidemiology and Immunity.

Noroviruses are major pathogens associated with acute gastroenteritis worldwide. Their RNA genomes are diverse, with two major genogroups (GI and GII) comprised of at least 28 genotypes associated with human disease. To elucidate mechanisms underlying norovirus diversity and evolution, we used a large-scale genomics approach to analyze human norovirus sequences. Comparison of over 2000 nearly full-length ORF2 sequences representing most of the known GI and GII genotypes infecting humans showed a limited number (≤5) of distinct intra-genotypic variants within each genotype, with the exception of GII.4. The non-GII.4 genotypes were comprised of one or more intra-genotypic variants, with each variant containing strains that differed by only a few residues over several decades (remaining "static") and that have co-circulated with no clear epidemiologic pattern. In contrast, the GII.4 genotype presented the largest number of variants (>10) that have evolved over time with a clear pattern of periodic variant replacement. To expand our understanding of these two patterns of diversification ("static" versus "evolving"), we analyzed using NGS the nearly full-length norovirus genome in healthy individuals infected with GII.4, GII.6 or GII.17 viruses in different outbreak settings. The GII.4 viruses accumulated mutations rapidly within and between hosts, while the GII.6 and GII.17 viruses remained relatively stable, consistent with their diversification patterns. Further analysis of genetic relationships and natural history patterns identified groupings of certain genotypes into larger related clusters designated here as "immunotypes". We propose that "immunotypes" and their evolutionary patterns influence the prevalence of a particular norovirus genotype in the human population.

Sequential Gastroenteritis Outbreaks in a Single Year Caused by Norovirus Genotypes GII.2 and GII.6 in an Institutional Setting.

BACKGROUND: Norovirus is a leading cause of acute gastroenteritis worldwide. Improved diagnostic capability has been instrumental in the characterization of archival norovirus strains associated with gastroenteritis outbreaks that were investigated decades ago. One such investigation was that of 2 sequential gastroenteritis outbreaks that occurred in 1971 at the former Henryton State Hospital in Maryland. Approximately 40% of the resident population experienced clinical symptoms in both outbreaks, which occurred 11 months apart. METHODS: Stored stools and paired sera were re-analyzed to investigate the etiology of the 2 outbreaks. RESULTS: Different norovirus genotypes were identified as the etiological agents responsible for the illnesses, with GII.2 associated with the first outbreak and GII.6 with the second. The viruses were antigenically distinct as determined by analyses of hyperimmune sera raised against the corresponding virus-like particles in animals, as well as paired sera from infected individuals. CONCLUSIONS: The observed antigenic differences were consistent with the failure of the GII.2 strain to provide cross-protective immunity to the GII.6 strain a few months later. An understanding of antigenic diversity among norovirus genotypes will be important in the design of norovirus vaccines.

Epidemiology of Norovirus Infection Among Immunocompromised Patients at a Tertiary Care Research Hospital, 2010-2013.

Background. Noroviruses are a major cause of infectious gastroenteritis worldwide, and viruses can establish persistent infection in immunocompromised individuals. Risk factors and transmission in this population are not fully understood. Methods. From 2010 through 2013, we conducted a retrospective review among immunocompromised patients (n = 268) enrolled in research studies at the National Institutes of Health Clinical Center and identified a subset of norovirus-positive patients (n = 18) who provided stool specimens for norovirus genotyping analysis. Results. Norovirus genome was identified by reverse-transcription quantitative polymerase chain reaction in stools of 35 (13%) of the 268 immunocompromised patients tested, and infection prevalence was 21% (11 of 53) in persons with primary immune deficiencies and 12% (20 of 166) among persons with solid tumors or hematologic malignancies. Among 18 patients with norovirus genotyping information, norovirus GII.4 was the most prevalent genotype (14 of 18, 78%). Persistent norovirus infection (≥6 months) was documented in 8 of 18 (44%) individuals. Phylogenetic analysis of the GII.4 capsid protein sequences identified at least 5 now-displaced GII.4 variant lineages, with no evidence of their nosocomial transmission in the Clinical Center. Conclusions. Norovirus was a leading enteric pathogen identified in this immunocompromised population. Both acute and chronic norovirus infections were observed, and these were likely community-acquired. Continued investigation will further define the role of noroviruses in these patients and inform efforts toward prevention and treatment.