Gammaherpesviruses are ubiquitous oncogenic pathogens that establish lifelong latency, are associated with multiple types of malignancies, and are increasingly believed to play a role in autoimmune disease. Human Epstein–Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) frequently infect across various stages of childhood development. Nevertheless, how infection during the earliest stages of life, when immune defenses are still developing, shapes the risk of malignant disease and autoimmunity remains poorly understood. Here, we characterized acute and chronic infection following early-life inoculation with murine gammaherpesvirus 68 (MHV68). We infected mice at postnatal day P3, P5, or P8, ages that, in the compressed developmental timeline of the mouse model, correspond to the progressive immune maturation that occurs in human neonatal to infancy stages. Mice infected during early life displayed age- and dose-dependent susceptibility to MHV68 infection, with the approximate lethal dose 50% (LD50) values ranging from 200 plaque forming units (PFU) in mice inoculated at P3 to 10,000 PFU in mice inoculated at P8. Severe disease was accompanied by substantial weight loss prior to death. Following intranasal inoculation of P8 pups, MHV68 replicated rapidly in the lung, reaching high titers by 3 days, achieving higher lung titers than adult mice at 5 days. Nevertheless, acute replication was largely resolved by 16 days in surviving animals. Early-life infection induced splenomegaly and resulted in the establishment of latency at frequencies similar to those of adult mice. In contrast, mice infected as neonates maintained 14-fold higher frequencies of latently infected cells than adults during the long-term stable phase of latency, months after clearance of a lytic infection, suggesting that early-life infection substantially alters the long-term latent reservoir. Together, these data demonstrate for the first time that gammaherpesvirus exposure during early life imprints a long-lasting increase in latency burden and establishes a foundation to determine potential implications for malignancy and autoimmune risk later in life.

Neonatal bile acid metabolism is distinct from that of adults due to developmental regulation of key transporters and enzymes. The apical sodium-dependent bile acid transporter (ASBT) is transiently repressed in the intestine after birth, yet its role in neonatal bile acid homeostasis remains unclear. Here, we demonstrate that ASBT plays a crucial role in limiting fecal bile acid loss and suppressing hepatic bile acid synthesis in neonates. ASBT-deficient pups exhibited a marked decrease in serum bile acids and concomitant increase in fecal bile acids, accompanied by upregulated hepatic bile acid synthesis genes, including CYP7A1, CYP7B1, and CYP27A1. We also illuminated a tissue-specific distinction in neonatal negative feedback regulation of bile acid synthesis, with intact hepatic regulation but impaired intestinal regulation. Our study identifies ASBT as a key regulator of neonatal bile acid homeostasis despite its strong repression early in life, highlighting its role in bile acid retention and synthesis regulation.NEW & NOTEWORTHY Despite being repressed after birth, ASBT is essential for neonatal bile acid homeostasis. This study reveals that ASBT limits fecal bile acid loss and suppresses hepatic bile acid synthesis in neonates. ASBT-deficient pups showed reduced serum bile acids, increased fecal loss, and upregulation of bile acid synthesis genes. Notably, feedback regulation of bile acid synthesis was intact in the liver but impaired in the intestine, uncovering tissue-specific control mechanisms in early life.

Maternal diet is critical in shaping neonatal metabolism and long-term health by governing breast milk composition. Although bile acids are present in breast milk, their functional role in infant development is not well understood. We identify enteromammary trafficking as the primary source of milk bile acids and show that this pool is modifiable by maternal diet. We also find that maternal bile acids regulate infant growth and levels of the growth-promoting hormone insulin-like growth factor 1 (IGF-1). Remarkably, maternal bile acid sequestration completely prevents excess weight gain in offspring nursed by dams on a high-fat diet. Supplementation with a bile acid or an agonist of the bile acid receptor TGR5 restores growth. Furthermore, TGR5-deficient pups phenocopy the maternal sequestration phenotype, supporting the model that maternal milk bile acids activate neonatal TGR5 to promote infant growth. Altogether, these findings reveal milk bile acids as active metabolic signals with potential for nutritional intervention in early-life programming.

Microbiota and feeding modes influence the susceptibility of premature newborns to necrotizing enterocolitis (NEC) through mechanisms that remain unknown. Here, we show that microbiota colonization facilitated by breastmilk feeding promotes NOD-like receptor family CARD domain containing 5 (Nlrc5) gene expression in mouse intestinal epithelial cells (IECs). Notably, inducible knockout of the Nlrc5 gene in IECs predisposes neonatal mice to NEC-like injury in the small intestine upon viral inflammation in an NK1.1+ cell-dependent manner. By contrast, formula feeding enhances neonatal gut colonization with environment-derived tilivalline-producing Klebsiella spp. Remarkably, tilivalline disrupts microbiota-activated STAT1 signaling that controls Nlrc5 gene expression in IECs through a PPAR-γ-mediated mechanism. Consequently, this dysregulation hinders the resistance of neonatal intestinal epithelium to self-NK1.1+ cell cytotoxicity upon virus infection/colonization, promoting NEC development. Together, we discover the underappreciated role of intestinal microbiota colonization in shaping a disease tolerance program to viral inflammation and elucidate the mechanisms impacting NEC development in neonates.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

The pathogenic outcome of enteric virus infections is governed by a complex interplay between the virus, intestinal microbiota, and host immune factors, with metabolites serving as a key mediator. Noroviruses bind bile acid metabolites, which are produced by the host and then modified by commensal bacteria. Paradoxically, bile acids can have both proviral and antiviral roles during norovirus infections. Working in an infant mouse model of norovirus infection, we demonstrate that microbiota and their bile acid metabolites protect from norovirus diarrhea, whereas host bile acids promote disease. We also find that maternal bile acid metabolism determines the susceptibility of newborn mice to norovirus diarrhea during breastfeeding. Finally, targeting maternal and neonatal bile acid metabolism can protect newborn mice from norovirus disease. In summary, neonatal metabolic immaturity and breastmilk bile acids are central determinants of heightened newborn vulnerability to norovirus disease.

Murine norovirus (MNV) is a positive-sense, plus-stranded RNA virus in the Caliciviridae family. Viruses in this family replicate in the intestine and are transmitted by the fecal-oral route. MNV is related to the human noroviruses, which cause the majority of nonbacterial gastroenteritis worldwide. Given the technical challenges in studying human norovirus, MNV is often used to study mechanisms in norovirus biology since it combines the availability of a cell culture and reverse genetics system with the ability to study infection in the native host. Adding to our previous protocol collection, here we describe additional techniques that have since been developed to study MNV biology. (c) 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Indirect method for measuring cell cytotoxicity and antiviral activityBasic Protocol 2: Measuring murine norovirus genome titers by RT-qPCRSupport Protocol 1: Preparation of standardBasic Protocol 3: Generation of recombinant murine norovirus with minimal passagingBasic Protocol 4: Generation of recombinant murine norovirus via circular polymerase extension reaction (CPER)Basic Protocol 5: Expression of norovirus NS1-2 in insect cell suspension cultures using a recombinant baculovirusSupport Protocol 2: Isotope labelling of norovirus NS1-2 in insect cellsSupport Protocol 3: Purification of the norovirus NS1-2 proteinSupport Protocol 4: Expression of norovirus NS1-2 in mammalian cells by transduction with a recombinant baculovirusBasic Protocol 6: Infection of enteroids in transwell inserts with murine norovirusSupport Protocol 5: Preparation of conditioned medium for enteroids cultureSupport Protocol 6: Isolation of crypts for enteroids generationSupport Protocol 7: Enteroid culture passaging and maintenanceBasic Protocol 7: Quantification of murine norovirus-induced diarrhea using neonatal mouse infectionsAlternate Protocol 1: Intragastric inoculation of neonatal miceAlternate Protocol 2: Scoring colon contents

Noroviruses are the leading cause of severe childhood diarrhea and foodborne disease worldwide. While they are a major cause of disease in all age groups, infections in the very young can be quite severe, with annual estimates of 50,000–200,000 fatalities in children under 5 years old. In spite of the remarkable disease burden associated with norovirus infections, very little is known about the pathogenic mechanisms underlying norovirus diarrhea, principally because of the lack of tractable small animal models. The development of the murine norovirus (MNV) model nearly two decades ago has facilitated progress in understanding host–norovirus interactions and norovirus strain variability. However, MNV strains tested thus far either do not cause intestinal disease or were isolated from extraintestinal tissue, raising concerns about translatability of research findings to human norovirus disease. Consequently, the field lacks a strong model of norovirus gastroenteritis. Here we provide a comprehensive characterization of a new small animal model system for the norovirus field that overcomes prior weaknesses. Specifically, we demonstrate that the WU23 MNV strain isolated from a mouse naturally presenting with diarrhea causes a transient reduction in weight gain and acute self-resolving diarrhea in neonatal mice of several inbred mouse lines. Moreover, our findings reveal that norovirus-induced diarrhea is associated with infection of subepithelial cells in the small intestine and systemic spread. Finally, type I interferons (IFNs) are critical to protect hosts from norovirus-induced intestinal disease whereas type III IFNs exacerbate diarrhea. This latter finding is consistent with other emerging data implicating type III IFNs in the exacerbation of some viral diseases. This new model system should enable a detailed investigation of norovirus disease mechanisms.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals.

Norovirus (NoV) is the leading global cause of viral gastroenteritis. Young children bear the highest burden of disease and play a key role in viral transmission throughout the population. However, which host factors contribute to age-associated variability in NoV severity and shedding are not well-defined. The murine NoV (MNoV) strain CR6 causes persistent infection in adult mice and targets intestinal tuft cells. Here we find that natural transmission of CR6 from infected dams occurred only in juvenile mice. Direct oral CR6 inoculation of wild-type neonatal mice led to accumulation of viral RNA in the ileum and prolonged shedding in the stool that was replication-independent. This viral exposure induced both innate and adaptive immune responses including interferon-stimulated gene expression and MNoV-specific antibody responses. Interestingly, viral uptake depended on passive ileal absorption of luminal virus, a process blocked by cortisone acetate administration, which prevented ileal viral RNA accumulation. Neonates lacking interferon signalling in haematopoietic cells were susceptible to productive infection, viral dissemination and lethality, which depended on the canonical MNoV receptor CD300LF. Together, our findings reveal developmentally associated aspects of persistent MNoV infection, including distinct tissue and cellular tropism, mechanisms of interferon regulation and severity of infection in the absence of interferon signalling. These emphasize the importance of defining viral pathogenesis phenotypes across the developmental spectrum and highlight passive viral uptake as an important contributor to enteric infections in early life.

As the largest mucosal surface, the gastrointestinal (GI) tract plays a key role in protecting the host against pathogen infections. It is a first line of defense against enteric viruses and must act to control infection while remaining tolerant to the high commensal bacteria load found within the GI tract. The GI tract can be divided into six main sections (stomach, duodenum, jejunum, ileum, colon, and rectum), and enteric pathogens have evolved to infect distinct parts of the GI tract. The intestinal epithelial cells (IECs) lining the GI tract are immune competent and can counteract these infections through their intrinsic immune response. Type I and type III interferons (IFNs) are antiviral cytokines that play a key role in protecting IECs against viruses with the type III IFN being the most important. Recent work has shown that IECs derived from the different sections of the GI tract display a unique expression of pattern recognition receptors used to fight pathogen infections. Additionally, it was also shown that these cells show a section-specific response to enteric viruses. This mini-review will discuss the molecular strategies used by IECs to detect and combat enteric viruses highlighting the differences existing along the entero-caudal axis of the GI tract. We will provide a perspective on how these spatially controlled mechanisms may influence virus tropism and discuss how the intestinal micro-environment may further shape the response of IECs to virus infections.

Human norovirus (HNoV) is a global health and socio-economic burden, estimated to infect every individual at least five times during their lifetime. The underlying mechanism for the potential lack of long-term immune protection from HNoV infections is not understood and prompted us to investigate HNoV susceptibility of primary human B cells and its functional impact. Primary B cells isolated from whole-blood were infected with HNoV-positive stool samples and harvested 3 days post infection (dpi) to assess viral RNA yield by RT-qPCR. A 3-18 fold increase in HNoV RNA yield was observed in 50-60% donors. Infection was further confirmed in B cells derived from splenic and lymph node biopsies. Next, we characterized infection of whole-blood derived B cells by flow cytometry in specific functional B cell subsets (naïve CD27-IgD+, memory switched CD27+IgD-, memory unswitched CD27+IgD+ and double-negative CD27-IgD-). While susceptibility of subsets was similar, we observed changes in B cell subsets distribution upon infection that were recapitulated after treatment with HNoV virus-like particles and mRNA encoding for HNoV NS1-2 protein. Importantly, treatment of immortalized BJAB B cell lines with the predicted recombinant NS1 protein triggered cell proliferation, increased ATP production, and induced metabolic changes, as detected by means of CFSE/Ki67 staining, seahorse analysis and metabolomics, respectively. These data demonstrate the susceptibility of primary B cells to HNoV infection and suggest that the secreted NS1 protein affects B cell function, proliferation and metabolism in vitro , which could have implications for viral pathogenesis and immune response in vivo .Importance Human norovirus (HNoV) is the most prevalent causative agent of gastroenteritis worldwide. Infection results in a self-limiting disease that can become chronic and severe in the immunocompromised, elderly and infants. There are currently no approved therapeutic and preventative strategies to limit the health and socio-economic burden associated with HNoV infections. Moreover, HNoV does not elicit life-long immunity as repeat infections are common, presenting a challenge for vaccine development. Given the importance of B cells for humoral immunity, we investigated susceptibility and impact of HNoV infection on human B cells. We found that HNoV replicates in human primary B cells derived from blood, spleen and lymph nodes specimens and induces functional changes in B cells, mediated in part by the non-structural protein NS1. Because of the secreted nature of NS1, we put forward the hypothesis that HNoV infection can modulate bystander B cell function with potential implications in systemic immune response.

Background: The COVID-19 pandemic has resulted in 275 million infections and 5.4 million deaths as of December 2021. While effective vaccines are being administered globally, there is still a great need for antiviral therapies as antigenically novel SARS-CoV-2 variants continue to emerge across the globe. Viruses require host factors at every step in their life cycle, representing a rich pool of candidate targets for antiviral drug design. Methods: To identify host factors that promote SARS-CoV-2 infection with potential for broad-spectrum activity across the coronavirus family, we performed genome-scale CRISPR knockout screens in two cell lines (Vero E6 and HEK293T ectopically expressing ACE2) with SARS-CoV-2 and the common cold-causing human coronavirus OC43. Gene knockdown, CRISPR knockout, and small molecule testing in Vero, HEK293, and human small airway epithelial cells were used to verify our findings. Results: While we identified multiple genes and functional pathways that have been previously reported to promote human coronavirus replication, we also identified a substantial number of novel genes and pathways. The website https://sarscrisprscreens.epi.ufl.edu/ was created to allow visualization and comparison of SARS-CoV2 CRISPR screens in a uniformly analyzed way. Of note, host factors involved in cell cycle regulation were enriched in our screens as were several key components of the programmed mRNA decay pathway. The role of EDC4 and XRN1 in coronavirus replication in human small airway epithelial cells was verified. Finally, we identified novel candidate antiviral compounds targeting a number of factors revealed by our screens. Conclusions: Overall, our studies substantiate and expand the growing body of literature focused on understanding key human coronavirus-host cell interactions and exploit that knowledge for rational antiviral drug development.

Noroviruses are the leading cause of severe childhood diarrhea and foodborne disease worldwide. While they are a major cause of disease in all age groups, infections in the very young can be quite severe with annual estimates of 50,000-200,000 fatalities in children under 5 years old. In spite of the remarkable disease burden associated with norovirus infections in people, very little is known about the pathogenic mechanisms underlying norovirus diarrhea, principally because of the lack of tractable small animal models. We recently demonstrated that wild-type neonatal mice are susceptible to murine norovirus (MNV)-induced acute self-resolving diarrhea in a time course mirroring human norovirus disease. Using this robust pathogenesis model system, we demonstrate that virulence is regulated by the responsiveness of the viral capsid to environmental cues that trigger contraction of the VP1 protruding (P) domain onto the particle shell, thus enhancing receptor binding and infectivity. The capacity of a given MNV strain to undergo this contraction positively correlates with infection of cells expressing low abundance of the virus receptor CD300lf, supporting a model whereby virion contraction triggers infection of CD300lflo cell types that are responsible for diarrhea induction. These findings directly link environmentally-influenced biophysical features with norovirus disease severity.

Le tractus intestinal humain abrite un groupe très diversifié de microorganismes commensaux qui jouent un rôle important dans l’homéostasie et la santé de l’intestin. Ce microbiote intestinal est essentiel au bon développement du système immunitaire de l’hôte, au métabolisme normal de l’intestin, à l’absorption des nutriments, à la protection contre les espèces bactériennes envahissantes et au maintien d’une physiologie intestinale normale.

Intestinal microbiota have profound effects on viral infections locally and systemically. While they can directly influence enteric virus infections, there is also an increasing appreciation for the role of microbiota-derived metabolites in regulating virus infections. Because metabolites diffuse across the intestinal epithelium and enter circulation, they can influence host response to pathogens at extraintestinal sites. In this review, we summarize the effects of three types of microbiota-derived metabolites on virus infections. While short-chain fatty acids serve to regulate the extent of inflammation associated with viral infections, the flavonoid desaminotyrosine and bile acids generally regulate interferon responses. A common theme that emerges is that microbiota-derived metabolites can have proviral and antiviral effects depending on the virus in question. Understanding the molecular mechanisms by which microbiota-derived metabolites impact viral infections and the highly conditional nature of these responses should pave the way to developing novel rational antivirals.

Interferons (IFNs) are key controllers of viral replication, with intact IFN responses suppressing virus growth and spread. Using the murine norovirus (MNoV) system, we show that IFNs exert selective pressure to limit the pathogenic evolutionary potential of this enteric virus. In animals lacking type I IFN signaling, the nonlethal MNoV strain CR6 rapidly acquired enhanced virulence via conversion of a single nucleotide. This nucleotide change resulted in amino acid substitution F514I in the viral capsid, which led to >10,000-fold higher replication in systemic organs including the brain. Pathogenicity was mediated by enhanced recruitment and infection of intestinal myeloid cells and increased extraintestinal dissemination of virus. Interestingly, the trade-off for this mutation was reduced fitness in an IFN-competent host, in which CR6 bearing F514I exhibited decreased intestinal replication and shedding. In an immunodeficient context, a spontaneous amino acid change can thus convert a relatively avirulent viral strain into a lethal pathogen.