Single-cell transcriptional analysis of murine norovirus infection in a human intestinal cell line.

Noroviruses are a major agent of acute gastroenteritis in humans, but host cell requirements for efficient replication have not been established. We engineered a human intestinal cell line (designated mCD300lf-hCaco2) expressing the murine norovirus (MNV) receptor, mouse CD300lf to become fully permissive for MNV replication. To explore the replicative machinery and host response of these cells, we performed a single-cell RNA sequencing (scRNA-seq) transcriptomics analysis of an MNV infection over time.

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.

Reconstitution of norovirus-specific T cell responses following hematopoietic stem cell transplantation in patients with inborn errors of immunity and chronic norovirus infection.

Background: Chronic norovirus infection (CNI) causes significant morbidity in immunocompromised patients. No effective prevention or treatment currently exists.

Methods: Two patients with inborn errors of immunity, X- linked severe combined immunodeficiency (X-SCID) and DOCK8 deficiency, were followed longitudinally for clinical course, immune reconstitution, norovirus-specific T cell (NST) response, B cell reconstitution, and norovirus-specific antibody production.

A non-human primate model for human norovirus infection.

Norovirus infection can cause gastrointestinal disease in humans. Development of therapies and vaccines against norovirus have been limited by the lack of a suitable and reliable animal model. Here we established rhesus macaques as an animal model for human norovirus infection.

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

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.

A single nanobody neutralizes multiple epochally evolving human noroviruses by modulating capsid plasticity.

Acute gastroenteritis caused by human noroviruses (HuNoVs) is a significant global health and economic burden and is without licensed vaccines or antiviral drugs. The GII.4 HuNoV causes most epidemics worldwide.

A luciferase-based approach for measuring HBGA blockade antibody titers against human norovirus.

Background: Noroviruses are the most common cause of viral gastroenteritis worldwide, yet there is a deficit in the understanding of protective immunity. Surrogate neutralization assays have been widely used that measure the ability of antibodies to block virus-like particle (VLP) binding to histo-blood group antigens (HBGAs). However, screening large sample sets against multiple antigens using the traditional HBGA blocking assay requires significant investment in terms of time, equipment, and technical expertise, largely associated with the generation of purified VLPs.

Generation of Norovirus-Specific T Cells From Human Donors With Extensive Cross-Reactivity to Variant Sequences: Implications for Immunotherapy.

Background: Chronic norovirus infection in immunocompromised patients can be severe, and presently there is no effective treatment. Adoptive transfer of virus-specific T cells has proven to be safe and effective for the treatment of many viral infections, and this could represent a novel treatment approach for chronic norovirus infection. Hence, we sought to generate human norovirus-specific T cells (NSTs) that can recognize different viral sequences.

Detection of Human Norovirus-Specific Antibodies Using the Luciferase Immunoprecipitation System (LIPS) Assay.

The luciferase immunoprecipitation system (LIPS) assay is a liquid-phase immunoassay that quantitates antigen-specific serum antibodies by measuring luminescence emitted by the reporter enzyme Renilla luciferase (Ruc) fused to an antigen of interest. The LIPS assay can be utilized as a high-throughput and sensitive serological method for profiling serum antibodies recognizing diverse antigens. This chapter provides a detailed protocol for detecting human norovirus-specific serum antibodies with the LIPS assay.

Vesicle-Cloaked Virus Clusters Are Optimal Units for Inter-organismal Viral Transmission.

In enteric viral infections, such as those with rotavirus and norovirus, individual viral particles shed in stool are considered the optimal units of fecal-oral transmission. We reveal that rotaviruses and noroviruses are also shed in stool as viral clusters enclosed within vesicles that deliver a high inoculum to the receiving host. Cultured cells non-lytically release rotaviruses and noroviruses inside extracellular vesicles.

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 BALB/c-derived RAW264.

A Luciferase Immunoprecipitation System (LIPS) assay for profiling human norovirus antibodies.

A luciferase immunoprecipitation systems (LIPS) assay was developed to define the antigenic specificity and titer of antibodies directed against human norovirus (HuNoV). Recombinant proteins, expressed by plasmid constructs encoding Renilla luciferase (Ruc) fused to the full-length HuNoV major capsid protein (VP1) (Ruc-antigen), were generated for ten HuNoV strains. In addition, subdomain constructs Ruc-Shell (S) and Ruc-Protruding (P) were engineered for a representative GII.

Human Sera Collected between 1979 and 2010 Possess Blocking-Antibody Titers to Pandemic GII.4 Noroviruses Isolated over Three Decades.

The emergence of pandemic GII.4 norovirus (NoV) strains has been proposed to occur due to changes in receptor usage and thereby to lead to immune evasion. To address this hypothesis, we measured the ability of human sera collected between 1979 and 2010 to block glycan binding of four pandemic GII.

Identification of Human Junctional Adhesion Molecule 1 as a Functional Receptor for the Hom-1 Calicivirus on Human Cells.

The Hom-1 vesivirus was reported in 1998 following the inadvertent transmission of the animal calicivirus San Miguel sea lion virus to a human host in a laboratory. We characterized the Hom-1 strain and investigated the mechanism by which human cells could be infected. An expression library of 3,559 human plasma membrane proteins was screened for reactivity with Hom-1 virus-like particles, and a single interacting protein, human junctional adhesion molecule 1 (hJAM1), was identified.

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.

The Norovirus NS3 Protein Is a Dynamic Lipid- and Microtubule-Associated Protein Involved in Viral RNA Replication.

Unlabelled: Norovirus (NoV) infections are a significant health burden to society, yet the lack of reliable tissue culture systems has hampered the development of appropriate antiviral therapies. Here we show that the NoV NS3 protein, derived from murine NoV (MNV), is intimately associated with the MNV replication complex and the viral replication intermediate double-stranded RNA (dsRNA). We observed that when expressed individually, MNV NS3 and NS3 encoded by human Norwalk virus (NV) induced the formation of distinct vesicle-like structures that did not colocalize with any particular protein markers to cellular organelles but localized to cellular membranes, in particular those with a high cholesterol content.

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

 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.  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.

Mapping and modeling of a strain-specific epitope in the Norwalk virus capsid inner shell.

Noroviruses are diverse positive-strand RNA viruses associated with acute gastroenteritis. Cross-reactive epitopes have been mapped primarily to conserved sequences in the capsid VP1 Shell (S) domain, and strain-specific epitopes to the highly variable Protruding (P) domain. In this work, we investigated a strain-specific linear epitope defined by MAb NV10 that was raised against prototype (Genogroup I.

Nucleolin promotes in vitro translation of feline calicivirus genomic RNA.

Feline calicivirus depends on host-cell proteins for its replication. We previously showed that knockdown of nucleolin (NCL), a phosphoprotein involved in ribosome biogenesis, resulted in the reduction of FCV protein synthesis and virus yield. Here, we found that NCL may not be involved in FCV binding and entry into cells, but it binds to both ends of the FCV genomic RNA, and stimulates its translation in vitro.

Llama nanoantibodies with therapeutic potential against human norovirus diarrhea.

Noroviruses are a major cause of acute gastroenteritis, but no vaccines or therapeutic drugs are available. Llama-derived single chain antibody fragments (also called VHH) are small, recombinant monoclonal antibodies of 15 kDa with several advantages over conventional antibodies. The aim of this study was to generate recombinant monoclonal VHH specific for the two major norovirus (NoV) genogroups (GI and GII) in order to investigate their potential as immunotherapy for the treatment of NoV diarrhea.

Recovery of murine norovirus and feline calicivirus from plasmids encoding EMCV IRES in stable cell lines expressing T7 polymerase.

Reverse genetics systems constitute one of the most important and powerful tools to study the molecular biology of viruses. We developed a new strategy for the recovery of murine norovirus from a single plasmid in which a bacteriophage T7 RNA polymerase (T7pol) promoter for transcription and an EMCV IRES for efficient translation were engineered immediately upstream of the viral genome. Infectious noroviruses were recovered following transfection of the newly designed plasmid into nonpermissive BHK-21 and HEK293T cell lines that were engineered to express T7pol constitutively.

Genetic characterization of feline calicivirus strains associated with varying disease manifestations during an outbreak season in Missouri (1995-1996).

Feline calicivirus (FCV) is a common cause of mild to severe upper respiratory tract disease (URTD) in cats. FCV strain 21223 was isolated from a kitten with severe pneumonia in a disease outbreak with unusually high mortality (35 %) that occurred in a Missouri feline colony in 1995-1996. Phylogenetic analysis of the genome sequence of strain 21223 indicated the emergence of a new FCV strain.

Identification of a Broadly Cross-Reactive Epitope in the Inner Shell of the Norovirus Capsid.

Noroviruses are major pathogens associated with acute gastroenteritis. They are diverse viruses, with at least six genogroups (GI-GVI) and multiple genotypes defined by differences in the major capsid protein, VP1. This diversity has challenged the development of broadly cross-reactive vaccines as well as efficient detection methods.