Production of infectious reporter murine norovirus by VP2 trans-complementation.

Human norovirus (HuNoV) causes gastroenteritis, a disease with no effective therapy or vaccine, and does not grow well in culture. Murine norovirus (MNV) easily replicates in cell cultures and small animals and has often been used as a model to elucidate the structural and functional characteristics of HuNoV. An MNV plasmid-based reverse genetics system was developed to produce the modified recombinant virus. In this study, we attempted to construct the recombinant virus by integrating a foreign gene into MNV ORF3, which encodes the minor structural protein VP2. Deletion of VP2 expression abolished infectious particles from MNV cDNA clones, and supplying exogenous VP2 to the cells rescued the infectivity of cDNA clones without VP2 expression. In addition, the coding sequence of C-terminal ORF3 was essential for cDNA clones compensated with VP2 to produce infectious particles. Furthermore, the recombinant virus with exogenous reporter genes in place of the dispensable region of ORF3 was propagated when VP2 was constitutively supplied. Our findings indicate that foreign genes can be transduced into the norovirus ORF3 region when VP2 is supplied and that successive propagation of modified recombinant norovirus could lead to the development of norovirus-based vaccines or therapeutics.IMPORTANCEIn this study, we revealed that some of the coding regions of ORF3 could be replaced by a foreign gene and infectious virus could be produced when VP2 was supplied. Propagation of this virus depended on VP2 being supplied in trans, indicating that this virus could infect only once. Our findings help to elucidate the functions of VP2 in the virus lifecycle and to develop other caliciviral vectors for recombinant attenuated live enteric virus vaccines or therapeutics tools.

Murine norovirus mutants adapted to replicate in human cells reveal a post-entry restriction.

RNA viruses lack proofreading in their RNA polymerases and therefore exist as genetically diverse populations. By exposing these diverse viral populations to selective pressures, viruses with mutations that confer fitness advantages can be enriched. To examine factors important for viral tropism and host restriction, we passaged murine norovirus (MNV) in a human cell line, HeLa cells, to select mutant viruses with increased fitness in non-murine cells. A major determinant of host range is expression of the MNV receptor CD300lf on mouse cells, but additional host factors may limit MNV replication in human cells. We found that viruses passaged six times in HeLa cells had enhanced replication compared with the parental virus. The passaged viruses had several mutations throughout the viral genome, which were primarily located in the viral non-structural coding regions. Although viral attachment was not altered for the passaged viruses, their replication was higher than the parental virus when the entry was bypassed, suggesting that the mutant viruses overcame a post-entry block in human cells. Three mutations in the viral NS1 protein were sufficient for enhanced post-entry replication in human cells. We found that the human cell-adapted MNV variants had reduced fitness in murine BV2 cells and infected mice, with reduced viral titers. These results suggest a fitness tradeoff, where increased fitness in a non-native host cell reduces fitness in a natural host environment. Overall, this work suggests that MNV tropism is determined by the presence of not only the viral receptor but also post-entry factors. IMPORTANCE: Viruses infect specific species and cell types, which is dictated by the expression of host factors required for viral entry as well as downstream replication steps. Murine norovirus (MNV) infects mouse cells, but not human cells. However, human cells expressing the murine CD300lf receptor support MNV replication, suggesting that receptor expression is a major determinant of MNV tropism. To determine whether other factors influence MNV tropism, we selected for variants with enhanced replication in human cells. We identified mutations that enhance MNV replication in human cells and demonstrated that these mutations enhance infection at a post-entry replication step. Therefore, MNV infection of human cells is restricted at both entry and post-entry stages. These results shed new light on factors that influence viral tropism and host range.

Amino acid substitutions in norovirus VP1 dictate host dissemination via variations in cellular attachment.

IMPORTANCE: All viruses initiate infection by utilizing receptors to attach to target host cells. These virus-receptor interactions can therefore dictate viral replication and pathogenesis. Understanding the nature of virus-receptor interactions could also be important for the development of novel therapies. Noroviruses are non-enveloped icosahedral viruses of medical importance. They are a common cause of acute gastroenteritis with no approved vaccine or therapy and are a tractable model for studying fundamental virus biology. In this study, we utilized the murine norovirus model system to show that variation in a single amino acid of the major capsid protein alone can affect viral infectivity through improved attachment to suspension cells. Modulating plasma membrane mobility reduced infectivity, suggesting an importance of membrane mobility for receptor recruitment and/or receptor conformation. Furthermore, different substitutions at this site altered viral tissue distribution in a murine model, illustrating how in-host capsid evolution could influence viral infectivity and/or immune evasion.

Interactions between human norovirus and intestinal microbiota/microbes: A scoping review.

Human norovirus (HuNoV) is an important foodborne virus, which causes non-bacterial acute gastroenteritis and is associated with a high disease burden. Recently, researchers have focus on the interaction between HuNoV and intestinal microbiota/microbes and engaged in studies investigating the implications of this interaction on HuNoV infection. However, the interaction mechanism and the implication of this interaction on host remain obscure. Current scoping review aimed to systematically investigate the interaction between HuNoV and intestinal microbiota, as well as their implication on HuNoV or HuNoV related symptoms. We found that HuNoV could bind to intestinal microbes and affect the intestinal microbial composition, diversity, and microbial gene expression. In reverse, intestinal microbes could affect HuNoV infectivity, although demonstrating contradictory effects (i.e., promote or inhibit HuNoV replication). These contradictory effects existed among microbes, in part, could be attributed to the differences among microbes (histo-blood group antigens and/or other small molecule substances). Results of current scoping review could assist in the selection and isolation of potential microbial candidates to prevent and/or alleviate HuNoV related symptoms.

A Reliable Multifaceted Solution against Foodborne Viral Infections: The Case of RiLK1 Decapeptide.

Food-borne transmission is a recognized route for many viruses associated with gastrointestinal, hepatic, or neurological diseases. Therefore, it is essential to identify new bioactive compounds with broad-spectrum antiviral activity to exploit innovative solutions against these hazards. Recently, antimicrobial peptides (AMPs) have been recognized as promising antiviral agents. Indeed, while the antibacterial and antifungal effects of these molecules have been widely reported, their use as potential antiviral agents has not yet been fully investigated. Herein, the antiviral activity of previously identified or newly designed AMPs was evaluated against the non-enveloped RNA viruses, hepatitis A virus (HAV) and murine norovirus (MNV), a surrogate for human norovirus. Moreover, specific assays were performed to recognize at which stage of the viral infection cycle the peptides could function. The results showed that almost all peptides displayed virucidal effects, with about 90% of infectivity reduction in HAV or MNV. However, the decapeptide RiLK1 demonstrated, together with its antibacterial and antifungal properties, a notable reduction in viral infection for both HAV and MNV, possibly through direct interaction with viral particles causing their damage or hindering the recognition of cellular receptors. Hence, RiLK1 could represent a versatile antimicrobial agent effective against various foodborne pathogens including viruses, bacteria, and fungi.

Protective Effect of Select Bacterial Species Representative of Fresh Produce on Human Norovirus Surrogates Exposed to Disinfecting Pulsed Light.

Contamination of berries and leafy greens with human norovirus (HuNoV) is a major cause of outbreaks of epidemic gastroenteritis worldwide. Using murine norovirus type 1 (MNV-1) and Tulane virus, we studied the possible extension of HuNoV persistence by biofilm-producing epiphytic bacteria on fresh produce. Nine bacterial species frequently found on the surface of berries and leafy greens (Bacillus cereus, Enterobacter cloacae, Escherichia coli, Kocuria kristinae, Lactobacillus plantarum, Pantoea agglomerans, Pseudomonas fluorescens, Raoultella terrigena, and Xanthomonas campestris) were evaluated for the ability to form biofilms in the MBEC Assay Biofilm Inoculator and in 96-well microplates. The biofilm-forming bacteria were further tested for binding MNV-1 and Tulane virus and the ability to protect them against loss of capsid integrity upon exposure to disinfecting pulsed light at a fluence of 11.52 J/cm2. Based on viral reductions, MNV-1 did not benefit from attachment to biofilm whereas Tulane virus was significantly more resistant than the control when attached to biofilms of E. cloacae (P ≤ 0.01), E. coli (P ≤ 0.01), K. kristinae (P ≤ 0.01), P. agglomerans (P ≤ 0.05), or P. fluorescens (P ≤ 0.0001). Enzymatic dispersion of biofilm and microscopic observations suggest that the biofilm matrix composition may contribute to the virus resistance. Our results indicate that direct virus-biofilm interaction protects Tulane virus against disinfecting pulsed light, and that HuNoV on fresh produce therefore might resist such treatment more than suggested by laboratory tests so far. IMPORTANCE Recent studies have shown that bacteria may be involved in the attachment of HuNoV to the surface of fresh produce. Because these foods are difficult to disinfect by conventional methods without compromising product quality, nonthermal nonchemical disinfectants such as pulsed light are being investigated. We seek to understand how HuNoV interacts with epiphytic bacteria, particularly with biofilms formed by bacterial epiphytes, with cells and extracellular polymeric substances, and to determine if it thus escapes inactivation by pulsed light. The results of this study should advance understanding of the effects of epiphytic biofilms on the persistence of HuNoV particle integrity after pulsed light treatment and thus guide the design of novel pathogen control strategies in the food industry.

Lipopolysaccharide Tolerance Enhances Murine Norovirus Reactivation: An Impact of Macrophages Mainly Evaluated by Proteomic Analysis.

Because of endotoxemia during sepsis (a severe life-threatening infection), lipopolysaccharide (LPS) tolerance (the reduced responses to the repeated LPS stimulation) might be one of the causes of sepsis-induced immune exhaustion (the increased susceptibility to secondary infection and/or viral reactivation). In LPS tolerance macrophage (twice-stimulated LPS, LPS/LPS) compared with a single LPS stimulation (N/LPS), there was (i) reduced energy of the cell in both glycolysis and mitochondrial activities (extracellular flux analysis), (ii) decreased abundance of the following proteins (proteomic analysis): (a) complex I and II of the mitochondrial electron transport chain, (b) most of the glycolysis enzymes, (c) anti-viral responses with Myxovirus resistance protein 1 (Mx1) and Ubiquitin-like protein ISG15 (Isg15), (d) antigen presentation pathways, and (iii) the down-regulated anti-viral genes, such as Mx1 and Isg15 (polymerase chain reaction). To test the correlation between LPS tolerance and viral reactivation, asymptomatic mice with and without murine norovirus (MNV) infection as determined in feces were tested. In MNV-positive mice, MNV abundance in the cecum, but not in feces, of LPS/LPS mice was higher than that in N/LPS and control groups, while MNV abundance of N/LPS and control were similar. Additionally, the down-regulated Mx1 and Isg15 were also demonstrated in the cecum, liver, and spleen in LPS/LPS-activated mice, regardless of MNV infection, while N/LPS more prominently upregulated these genes in the cecum of MNV-positive mice compared with the MNV-negative group. In conclusion, defects in anti-viral responses after LPS tolerance, perhaps through the reduced energy status of macrophages, might partly be responsible for the viral reactivation. More studies on patients are of interest.

Norovirus elimination on the surface of fresh foods.

Fresh foods like fruits, vegetables and shellfish are potential sources for viral infections such as human norovirus (NoV). Chemical treatment like chlorination is a well-known process for food pathogens and virus elimination. However, with the increase of the consumer demand for less toxic treatment, the use of natural antimicrobials like essential oils from spice or plants, fruit extracts, and cold pasteurization treatments (fermentation, irradiation, ozonation and high pressure) could be considered. The aim of this review is to discuss these technologies and their efficacy to eliminate NoV on the surface of fresh food.

Efficacy of washing produce in removing human coronavirus OC43 and murine norovirus.

AIMS: Fresh produce is often a vehicle for the transmission of foodborne pathogens such as human norovirus. Thus, it is recommended to wash the surface of produce before consumption, and one of the most common ways to wash produce is by rinsing under running tap water. This study determined the effectiveness of removal of human coronavirus-OC43 (HCoV-OC43), as a surrogate for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and murine norovirus-1 (MNV-1), as a surrogate for human norovirus, from contaminated lettuce, apples and cucumbers. METHODS AND RESULTS: The produce surfaces were artificially inoculated in conjunction with faecal material to represent natural contamination. Rinsing under tap water for 10 s at 40 ml/s removed 1.94 ± 0.44, 1.42 ± 0.00 and 1.42 ± 0.42 log of HCoV-OC43 from apple, cucumber and lettuce respectively. The same washing technique removed 1.77 ± 0.17, 1.42 ± 0.07 and 1.79 ± 0.14 log of MNV-1 from apple, cucumber and lettuce respectively. This washing technique was effective at reducing a significant amount of viral contamination, however, it was not enough to eliminate the entire contamination. There was no significant difference in the reduction of viral load between the two viruses, nor between the three surfaces tested in this study. CONCLUSIONS: Our data suggest that washing under tap water would be an efficient way of reducing the risk of foodborne viral transmission only if the level of contamination is less than 2 log PFU. SIGNIFICANCE AND IMPACT OF STUDY: This study demonstrates that running tap water was effective at reducing the amount of infectious HCoV-OC43 and MNV on produce surfaces, and washing produce continues to be an important task to perform prior to consumption to avoid infection by foodborne viruses, particularly for foods which are eaten raw.

Multiplex PCR method for the detection of human norovirus, Salmonella spp., Shigella spp., and shiga toxin producing Escherichia coli in blackberry, coriander, lettuce and strawberry.

A multiplex PCR method was developed for the simultaneous detection of murine norovirus (MNV-1) as a surrogate for human norovirus (HuNoV) GI and GII, Salmonella spp., Shigella spp., and Shiga toxin producing Escherichia coli (STEC) in fresh produce. The toxicity of the glycine buffer on bacterial pathogens viability was evaluated. The growth of each of the three pathogens (previously stressed) was evaluated at 35 and 41.5 °C in modified buffered peptone water (mBPW) and trypticase soy broth (TSB), supplemented with vancomycin, novobiocin and brilliant green at two concentration levels. The selected conditions for simultaneous enrichment were: 41.5 °C/mBPW/supplemented with 8 ppm vancomycin, 0.6 ppm novobiocin and 0.2 ppm brilliant green. The pathogens and aerobic plate count (APC) growth was evaluated in the enrichment of lettuce, coriander, strawberry and blackberry under the best enrichment conditions. Starting from 1 to 10 CFU/mL, Salmonella reached from 7.63 to 8.91, Shigella 6.81 to 7.76 and STEC 7.43 to 9.27 log CFU/mL. The population reached for the APC was 5.11-6.56 log CFU/mL. Simultaneous detection by PCR was done using designed primers targeting invA, ipaH, stx1 and stx2 genes, and MNV-1. The detection sensitivity was 10-100 PFU for the MNV-1 and 1-10 CFU for each pathogenic bacteria. This protocol takes 6 h for MNV-1 and 24 h for Salmonella spp., Shigella spp., and STEC detection from the same food portion. In total, 200 samples were analyzed from retail markets from Queretaro, Mexico. Two strawberry samples were positive for HuNoV GI and one lettuce sample was positive for STEC. In conclusion, the method developed in this study is capable of detecting HuNoV GI and GII, Salmonella spp., Shigella spp and STEC from the same fresh produce sample.

Detection and whole genome sequencing of murine norovirus in animal facility in Italy.

Viruses belonging to the genus Norovirus (NoV) of the family Caliciviridae are the major cause of acute viral gastroenteritis worldwide. NoVs are classified into 10 genogroups (GI-GX), and those belonging to the genogroup GV are able to infect several species of rodents. To evaluate the circulation of MNV among mice housed in an Italian facility, sampling was performed over two separate periods, in 2011, and 3 years later in 2014. During the two samplings, 75 fecal samples were collected from healthy mice housed in the animal facility and subjected to RT-PCR for viral detection. After the analysis, 41/75 animals (54.6%) resulted positive for the presence of MNV in feces. Nucleotide sequencing revealed the presence of two MNV variants co-circulating in both 2011 and 2014. One MNV strain was isolated on RAW264.7 cell line, and subjected to full genome sequencing. Our study showed that the murine noroviruses are widespread in the investigated animal facility, despite guidelines for animal care and maintenance. Full genome sequence analysis of the MNV strain described in this study showed a correlation with other strains circulating in Europe. Understanding the molecular epidemiology of this virus should give insight into its natural history and evolution in mice.

Guanylate-binding protein 2 orchestrates innate immune responses against murine norovirus and is antagonized by the viral protein NS7.

Noroviruses are the main causative agents of acute viral gastroenteritis, but the host factors that restrict their replication remain poorly identified. Guanylate-binding proteins (GBPs) are interferon (IFN)-inducible GTPases that exert broad antiviral activity and are important mediators of host defenses against viral infections. Here, we show that both IFN-γ stimulation and murine norovirus (MNV) infection induce GBP2 expression in murine macrophages. Results from loss- and gain-of-function assays indicated that GBP2 is important for IFN-γ-dependent anti-MNV activity in murine macrophages. Ectopic expression of MNV receptor (CD300lf) in human HEK293T epithelial cells conferred susceptibility to MNV infection. Importantly, GBP2 potently inhibited MNV in these human epithelial cells. Results from mechanistic dissection experiments revealed that the N-terminal G domain of GBP2 mediates these anti-MNV effects. R48A and K51A substitutions in GBP2, associated with loss of GBP2 GTPase activity, attenuated the anti-MNV effects of GBP2. Finally, we found that nonstructural protein 7 (NS7) of MNV co-localizes with GBP2 and antagonizes the anti-MNV activity of GBP2. These findings reveal that GBP2 is an important mediator of host defenses against murine norovirus.

Airborne Disinfection by Dry Fogging Efficiently Inactivates Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Mycobacteria, and Bacterial Spores and Shows Limitations of Commercial Spore Carriers.

Airborne disinfection of high-containment facilities before maintenance or between animal studies is crucial. Commercial spore carriers (CSC) coated with 106 spores of Geobacillus stearothermophilus are often used to assess the efficacy of disinfection. We used quantitative carrier testing (QCT) procedures to compare the sensitivity of CSC with that of surrogates for nonenveloped and enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), mycobacteria, and spores, to an aerosolized mixture of peroxyacetic acid and hydrogen peroxide (aPAA-HP). We then used the QCT methodology to determine relevant process parameters to develop and validate effective disinfection protocols (≥4-log10 reduction) in various large and complex facilities. Our results demonstrate that aPAA-HP is a highly efficient procedure for airborne room disinfection. Relevant process parameters such as temperature and relative humidity can be wirelessly monitored. Furthermore, we found striking differences in inactivation efficacies against some of the tested microorganisms. Overall, we conclude that dry fogging a mixture of aPAA-HP is highly effective against a broad range of microorganisms as well as material compatible with relevant concentrations. Furthermore, CSC are artificial bioindicators with lower resistance and thus should not be used for validating airborne disinfection when microorganisms other than viruses have to be inactivated.IMPORTANCE Airborne disinfection is not only of crucial importance for the safe operation of laboratories and animal rooms where infectious agents are handled but also can be used in public health emergencies such as the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. We show that dry fogging an aerosolized mixture of peroxyacetic acid and hydrogen peroxide (aPAA-HP) is highly microbicidal, efficient, fast, robust, environmentally neutral, and a suitable airborne disinfection method. In addition, the low concentration of dispersed disinfectant, particularly for enveloped viral pathogens such as SARS-CoV-2, entails high material compatibility. For these reasons and due to the relative simplicity of the procedure, it is an ideal disinfection method for hospital wards, ambulances, public conveyances, and indoor community areas. Thus, we conclude that this method is an excellent choice for control of the current SARS-CoV-2 pandemic.

Electrochemical investigations for COVID-19 detection-A comparison with other viral detection methods.

Virus-induced infection such as SARS-CoV-2 is a serious threat to human health and the economic setback of the world. Continued advances in the development of technologies are required before the viruses undergo mutation. The low concentration of viruses in environmental samples makes the detection extremely challenging; simple, accurate and rapid detection methods are in urgent need. Of all the analytical techniques, electrochemical methods have the established capabilities to address the issues. Particularly, the integration of nanotechnology would allow miniature devices to be made available at the point-of-care. This review outlines the capabilities of electrochemical methods in conjunction with nanotechnology for the detection of SARS-CoV-2. Future directions and challenges of the electrochemical biosensors for pathogen detection are covered including wearable and conformal biosensors, detection of plant pathogens, multiplexed detection, and reusable biosensors for on-site monitoring, thereby providing low-cost and disposable biosensors.

SARS-CoV-2 in environmental perspective: Occurrence, persistence, surveillance, inactivation and challenges.

The unprecedented global spread of the severe acute respiratory syndrome (SARS) caused by SARS-CoV-2 is depicting the distressing pandemic consequence on human health, economy as well as ecosystem services. So far novel coronavirus (CoV) outbreaks were associated with SARS-CoV-2 (2019), middle east respiratory syndrome coronavirus (MERS-CoV, 2012), and SARS-CoV-1 (2003) events. CoV relates to the enveloped family of Betacoronavirus (ßCoV) with positive-sense single-stranded RNA (+ssRNA). Knowing well the persistence, transmission, and spread of SARS-CoV-2 through proximity, the faecal-oral route is now emerging as a major environmental concern to community transmission. The replication and persistence of CoV in the gastrointestinal (GI) tract and shedding through stools is indicating a potential transmission route to the environment settings. Despite of the evidence, based on fewer reports on SARS-CoV-2 occurrence and persistence in wastewater/sewage/water, the transmission of the infective virus to the community is yet to be established. In this realm, this communication attempted to review the possible influx route of the enteric enveloped viral transmission in the environmental settings with reference to its occurrence, persistence, detection, and inactivation based on the published literature so far. The possibilities of airborne transmission through enteric virus-laden aerosols, environmental factors that may influence the viral transmission, and disinfection methods (conventional and emerging) as well as the inactivation mechanism with reference to the enveloped virus were reviewed. The need for wastewater epidemiology (WBE) studies for surveillance as well as for early warning signal was elaborated. This communication will provide a basis to understand the SARS-CoV-2 as well as other viruses in the context of the environmental engineering perspective to design effective strategies to counter the enteric virus transmission and also serves as a working paper for researchers, policy makers and regulators.

Effect of heat shock on murine norovirus replication in RAW264.7 cells.

Murine norovirus (MNV), is a prevalent pathogen of laboratory mice closely related to human norovirus (HuNoV), a contagious pathogen known to cause gastroenteritis worldwide; however, the mechanism of norovirus replication remains poorly understood. Both heat shock protein 90 (Hsp90) and heat shock protein 70 (Hsp70) play an important role in viral genome replication and viral gene expression. In this study, we first found that heat stress exerted a positive effect on the replication of MNV in the murine macrophage RAW264.7 cell line. Inhibition of Hsp70 and Hsp90 by the specific inhibitors, KNK437 and 17-AGG, respectively showed that Hsp70 and Hsp90 enhanced MNV genome replication and virion production. In addition, we found that KNK437 and 17-AGG could decrease the level of IL-1ß, IL-10, and TNF-α mRNA expression in MNV-infected cells. These data suggested that heat stress can positively regulate MNV replication, which advances our understanding of the molecular mechanism of MNV infection.

The Effect of GD1a Ganglioside-Expressing Bacterial Strains on Murine Norovirus Infectivity.

In this study, we investigated the impact of GD1a-expressing bacterial strains on the infectivity of murine norovirus (MNV). Eligible bacterial strains were screened from a sewage sample using flow cytometry, and their genetic sequences of 16S rRNA were determined. The enzyme-linked immunosorbent assay (ELISA) was employed to analyze the binding between bacteria and MNV particles, and the plaque assay was used to assess the effects of GD1a-positive and negative strains on MNV infectivity. The result from ELISA shows that MNV particles are able to bind to both GD1a-positive and negative bacterial strains, but the binding to the GD1a-positive strain is more significant. The infectivity assay result further shows that the MNV infectious titer declined with an increasing concentration of GD1a-positive bacteria. The addition of anti-GD1a antibody in the infectivity assay led to the recovery of the MNV infectious titer, further confirming that the binding between MNV particles and bacterial GD1a ganglioside compromises MNV infectivity. Our findings highlight the role indigenous bacteria may play in the lifecycle of waterborne enteric viruses as well as the potential of exploiting them for virus transmission intervention and water safety improvement.

Atomic Structure of the Murine Norovirus Protruding Domain and Soluble CD300lf Receptor Complex.

Human noroviruses are the leading cause of acute gastroenteritis in humans. Noroviruses also infect animals, such as cows, mice, cats, and dogs. How noroviruses bind and enter host cells is still incompletely understood. Recently, the type I transmembrane protein CD300lf was identified as the murine norovirus receptor, yet it is unclear how the virus capsid and receptor interact at the molecular level. In this study, we determined the X-ray crystal structure of the soluble CD300lf (sCD300lf) and the murine norovirus capsid protruding domain complex at a 2.05-Å resolution. We found that the sCD300lf-binding site is located on the topside of the protruding domain and involves a network of hydrophilic and hydrophobic interactions. sCD300lf locked nicely into a complementary cavity on the protruding domain that is additionally coordinated with a positive surface charge on sCD300lf and a negative surface charge on the protruding domain. Five of six protruding domain residues interacting with sCD300lf were maintained between different murine norovirus strains, suggesting that sCD300lf was capable of binding to a highly conserved pocket. Moreover, a sequence alignment with other CD300 paralogs showed that the sCD300lf-interacting residues were partially conserved in CD300ld but variable in other CD300 family members, consistent with previously reported infection selectivity. Overall, these data provide insights into how a norovirus engages a protein receptor and will be important for a better understanding of selective recognition and norovirus attachment and entry mechanisms.IMPORTANCE Noroviruses exhibit exquisite host range specificity due to species-specific interactions between the norovirus capsid protein and host molecules. Given this strict host range restriction, it has been unclear how the viruses are maintained within a species between relatively sporadic epidemics. While much data demonstrate that noroviruses can interact with carbohydrates, recent work has shown that expression of the protein CD300lf is both necessary and sufficient for murine norovirus infection of mice and binding of the virus to permissive cells. Importantly, the expression of this murine protein by human cells renders them fully permissive for murine norovirus infection, indicating that at least in this case, host range restriction is determined by molecular events that control receptor binding and entry. Defining the atomic-resolution interactions between the norovirus capsid protein and its cognate receptor is essential for a molecular understanding of host-range restriction and norovirus tropism.

Functional receptor molecules CD300lf and CD300ld within the CD300 family enable murine noroviruses to infect cells.

Norovirus is the leading cause of acute gastroenteritis worldwide. Since the discovery of human norovirus (HuNoV), an efficient and reproducible norovirus replication system has not been established in cultured cells. Although limited amounts of virus particles can be produced when the HuNoV genome is directly transfected into cells, the HuNoV cycle of infection has not been successfully reproduced in any currently available cell-culture system. Those results imply that the identification of a functional cell-surface receptor for norovirus might be the key to establishing a norovirus culture system. Using a genome-wide CRISPR/Cas9 guide RNA library, we identified murine CD300lf and CD300ld as functional receptors for murine norovirus (MNV). The treatment of susceptible cells with polyclonal antibody against CD300lf significantly reduced the production of viral progeny. Additionally, ectopic CD300lf expression in nonsusceptible cell lines derived from other animal species enabled MNV infection and progeny production, suggesting that CD300lf has potential for dictating MNV host tropism. Furthermore, CD300ld, which has an amino acid sequence in the N-terminal region of its extracellular domain that is highly homologous to that of CD300lf, also functions as a receptor for MNV. Our results indicate that direct interaction of MNV with two cell-surface molecules, CD300lf and CD300ld, dictates permissive noroviral infection.

Antiviral Effects of Houttuynia cordata Polysaccharide Extract on Murine Norovirus-1 (MNV-1)-A Human Norovirus Surrogate.

Houttuynia cordata is an herbal plant rich in polysaccharides and with several pharmacological activities. Human noroviruses (HuNoVs) are the most common cause of foodborne viral gastroenteritis throughout the world. In this study, H. cordata polysaccharide (HP), with a molecular weight of ~43 kDa, was purified from H. cordata water extract (HWE). The polysaccharide HP was composed predominantly of galacturonic acid, galactose, glucose, and xylose in a molar ratio of 1.56:1.49:1.26:1.11. Methylation and NMR analyses revealed that HP was a pectin-like acidic polysaccharide mainly consisting of α-1,4-linked GalpA, ß-1,4-linked Galp, ß-1,4-linked Glcp, and ß-1,4-linked Xylp residues. To evaluate the antiviral activity of H. cordata extracts, we compared the anti-norovirus potential of HP with HWE and ethanol extract (HEE) from H. cordata by plaque assay (plaque forming units (PFU)/mL) for murine norovirus-1 (MNV-1), a surrogate of HuNoVs. Viruses at high (8.09 log10 PFU/mL) or low (4.38 log10 PFU/mL) counts were mixed with 100, 250, and 500 µg/mL of HP, HWE or HEE and incubated for 30 min at room temperature. H. cordata polysaccharide (HP) was more effective than HEE in reducing MNV-1 plaque formation, but less effective than HWE. When MNV-1 was treated with 500 µg/mL HP, the infectivity of MNV-1 decreased to an undetectable level. The selectivity indexes of each sample were 1.95 for HEE, 5.74 for HP, and 16.14 for HWE. The results of decimal reduction time and transmission electron microscopic revealed that HP has anti-viral effects by deforming and inflating virus particles, thereby inhibiting the penetration of viruses in target cells. These findings suggest that HP might have potential as an antiviral agent in the treatment of viral diseases.