Mosaic RBD nanoparticles induce intergenus cross-reactive antibodies and protect against SARS-CoV-2 challenge.

Recurrent spillovers of α- and ß-coronaviruses (CoV) such as severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome-CoV, SARS-CoV-2, and possibly human CoV have caused serious morbidity and mortality worldwide. In this study, six receptor-binding domains (RBDs) derived from α- and ß-CoV that are considered to have originated from animals and cross-infected humans were linked to a heterotrimeric scaffold, proliferating cell nuclear antigen (PCNA) subunits, PCNA1, PCNA2, and PCNA3. They assemble to create a stable mosaic multivalent nanoparticle, 6RBD-np, displaying a ring-shaped disk with six protruding antigens, like jewels in a crown. Prime-boost immunizations with 6RBD-np in mice induced significantly high Ab titers against RBD antigens derived from α- and ß-CoV and increased interferon (IFN-γ) production, with full protection against the SARS-CoV-2 wild type and Delta challenges. The mosaic 6RBD-np has the potential to induce intergenus cross-reactivity and to be developed as a pan-CoV vaccine against future CoV spillovers.

Antiviral Activities of Asarones and Rhizomes of Acorus gramineus on Murine Norovirus.

Noroviruses (NVs) are a major cause of foodborne diseases worldwide. The rhizomes of Acorus gramineus (AGR) have been used as a traditional medicinal plant and a food additive. In this study, AGR and its bioactive components-α-asarone and ß-asarone-showed significant antiviral activities against murine NV (MNV) with pre-treatment, with more than two log reductions in viral plaques. They also demonstrated strong inhibition on binding to A- and O-type saliva by the recombinant P domain derived from human NV (HuNV) GII.4. Both α- and ß-asarones also inhibited the binding of the P domain to the receptor at 0.125-1 mM in a concentration-dependent manner and induced a marked reduction in Tm, suggesting that they may reduce structural stability and block receptor binding by the P domain. In simulated digestive conditions, the AGR extract, α-asarone, or ß-asarone further showed a significant reduction of MNV plaques by 1.5-2.8 logs. The asarones show a potential for development as a scaffold for anti-NV agents.

Mori Cortex Radicis extract inhibits human norovirus surrogate in simulated digestive conditions.

Norovirus is a major cause of acute gastroenteritis globally, resulting in enormous health and societal costs. In this study, the antiviral activities of Mori Cortex Radicis (MCR) extract and its bioactive flavonoids, morusin and kuwanon G, were tested against murine norovirus (MNV), a human norovirus surrogate, using plaque assay. The antiviral activity was confirmed in simulated digestive conditions, including simulated saliva fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF). Pre-treatment of MNV with MCR extract at 1000 µg/mL showed antiviral activity with a 1.1-log reduction. Morusin and kuwanon G also demonstrated a 1.0-log and 0.6-log reductions of MNV titers, respectively, at 100 µM. MCR extract at a concentration of 2 mg/mL in SSF, SGF, and SIF markedly reduced MNV titers by 1.8, 1.9, and 1.5 logs, respectively. Therefore, these data suggest that MCR extract can be used to control norovirus infectivity.

Comprehensive genomic analysis reveals virulence factors and antibiotic resistance genes in Pantoea agglomerans KM1, a potential opportunistic pathogen.

Pantoea agglomerans is a Gram-negative facultative anaerobic bacillus causing a wide range of opportunistic infections in humans including septicemia, pneumonia, septic arthritis, wound infections and meningitis. To date, the determinants of virulence, antibiotic resistance, metabolic features conferring survival and host-associated pathogenic potential of this bacterium remain largely underexplored. In this study, we sequenced and assembled the whole-genome of P. agglomerans KM1 isolated from kimchi in South Korea. The genome contained one circular chromosome of 4,039,945 bp, 3 mega plasmids, and 2 prophages. The phage-derived genes encoded integrase, lysozyme and terminase. Six CRISPR loci were identified within the bacterial chromosome. Further in-depth analysis showed that the genome contained 13 antibiotic resistance genes conferring resistance to clinically important antibiotics such as penicillin G, bacitracin, rifampicin, vancomycin, and fosfomycin. Genes involved in adaptations to environmental stress were also identified which included factors providing resistance to osmotic lysis, oxidative stress, as well as heat and cold shock. The genomic analysis of virulence factors led to identification of a type VI secretion system, hemolysin, filamentous hemagglutinin, and genes involved in iron uptake and sequestration. Finally, the data provided here show that, the KM1 isolate exerted strong immunostimulatory properties on RAW 264.7 macrophages in vitro. Stimulated cells produced Nitric Oxide (NO) and pro-inflammatory cytokines TNF-α, IL-6 and the anti-inflammatory cytokine IL-10. The upstream signaling for production of TNF-α, IL-6, IL-10, and NO depended on TLR4 and TLR1/2. While production of TNF-α, IL-6 and NO involved solely activation of the NF-κB, IL-10 secretion was largely dependent on NF-κB and to a lesser extent on MAPK Kinases. Taken together, the analysis of the whole-genome and immunostimulatory properties provided in-depth characterization of the P. agglomerans KM1 isolate shedding a new light on determinants of virulence that drive its interactions with the environment, other microorganisms and eukaryotic hosts.

Magnolia officinalis and Its Honokiol and Magnolol Constituents Inhibit Human Norovirus Surrogates.

Norovirus is a major cause of foodborne disease and nonbacterial gastroenteritis globally. This study evaluated the antiviral effects of Magnolia officinalis extract and its honokiol and magnolol constituents against human norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV) in vitro, and in model food systems. Pretreatment or cotreatment of M. officinalis extract at 1 mg/mL reduced MNV and FCV titers by 0.6-1.8 log. Honokiol and magnolol, which are the major polyphenols in the extract, showed significant antiviral effects against MNV and FCV. The virus-infected cells that were treated with M. officinalis extract exhibited significantly increased glutathione levels (p < 0.05). The extract, honokiol, and magnolol revealed ferric ion-reducing and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities in a dose-dependent manner. Furthermore, MNV and FCV titers were reduced by >1.6 log or to undetectable levels in apple, orange, and plum juices and by 0.9 and 1.6 log in milk, respectively, when they were treated with the extract at 5 mg/mL. Therefore, the present study suggests that M. officinalis extract can be used as an antiviral food material to control norovirus foodborne diseases.

Inhibitory Effects of Laminaria japonica Fucoidans Against Noroviruses.

Norovirus is the leading cause of nonbacterial foodborne disease outbreaks. Human noroviruses (HuNoVs) bind to histo-blood group antigens as the host receptor for infection. In this study, the inhibitory effects of fucoidans from brown algae, Laminaria japonica (LJ), Undaria pinnatifida and Undaria pinnatifida sporophyll, were evaluated against murine norovirus (MNoV), feline calicivirus (FCV) and HuNoV. Pretreatment of MNoV or FCV with the fucoidans at 1 mg/mL showed high antiviral activities, with 1.1 average log reductions of viral titers in plaque assays. They also showed significant inhibition on the binding of the P domains of HuNoV GII.4 and GII.17 to A- or O-type saliva and the LJ fucoidan was the most effective, reaching 54-72% inhibition at 1 mg/mL. In STAT1-/- mice infected with MNoV, oral administration of the LJ fucoidan, composed of mainly sulfated fucose and minor amounts of glucose and galactose, improved the survival rates of mice and significantly reduced the viral titers in their feces. Overall, these results provide the LJ fucoidan can be used to reduce NoV outbreaks.

Divalent cation-induced conformational changes of influenza virus hemagglutinin.

Divalent cations Cu2+ and Zn2+ can prevent the viral growth in mammalian cells during influenza infection, and viral titers decrease significantly on a copper surface. The underlying mechanisms include DNA damage by radicals, modulation of viral protease, M1 or neuraminidase, and morphological changes in viral particles. However, the molecular mechanisms underlying divalent cation-mediated antiviral activities are unclear. An unexpected observation of this study was that a Zn2+ ion is bound by Glu68 and His137 residues at the head regions of two neighboring trimers in the crystal structure of hemagglutinin (HA) derived from A/Thailand/CU44/2006. The binding of Zn2+ at high concentrations induced multimerization of HA and decreased its acid stability. The acid-induced conformational change of HA occurred even at neutral pH in the presence of Zn2+. The fusion of viral and host endosomal membranes requires substantial conformational changes in HA upon exposure to acidic pH. Therefore, our results suggest that binding of Zn2+ may facilitate the conformational changes of HA, analogous to that induced by acidic pH.

Antiviral Activities of Mulberry (Morus alba) Juice and Seed against Influenza Viruses.

Antiviral activities of Morus alba (MA) juice and seed were examined using time-of-addition plaque assays against influenza viruses, A/Brisbane/59/2007 (H1N1) (BR59), pandemic A/Korea/01/2009(H1N1) (KR01), A/Brisbane/10/2007(H3N2) (BR10), and B/Florida/4/2006 (FL04). MA juice (MAJ) showed much higher antiviral activity than MA seed (MAS). In the pre- and cotreatment of virus, MAJ showed antiviral effects against BR59, KR01, and FL04 in a dose-dependent manner. In particular, MAJ at 4% concentration exhibited 1.3 log inhibition in the pre- and cotreatment of the virus against FL04, a type B virus. However, little or weak inhibition was observed in the posttreatment of MAJ. GSH levels in the virus-infected cells were also examined. The decreased levels by the viral infection were restored significantly by the addition of MAJ. MAJ also exhibited significant DPPH radical scavenging and ferric ion-reducing activities in a dose-dependent manner. Cyanidin-3-rutinoside, the most abundant polyphenol compound of MAJ identified by LC-MS in this study, showed weak inhibitory effects against FL04 in the pretreatment, whereas gallic acid, a minor compound of MAJ, revealed significant antiviral effect. These results suggest that MAJ can be developed as a novel plant-derived antiviral against influenza viruses.

Nucleotide triphosphatase and RNA chaperone activities of murine norovirus NS3.

Modulation of RNA structure is essential in the life cycle of RNA viruses. Immediate replication upon infection requires RNA unwinding to ensure that RNA templates are not in intra- or intermolecular duplex forms. The calicivirus NS3, one of the highly conserved nonstructural (NS) proteins, has conserved motifs common to helicase superfamily 3 among six genogroups. However, its biological functions are not fully understood. In this study we report the oligomeric state and the nucleotide triphosphatase (NTPase) and RNA chaperone activities of the recombinant full-length NS3 derived from murine norovirus (MNV). The MNV NS3 has an Mg2+-dependent NTPase activity, and site-directed mutagenesis of the conserved NTPase motifs blocked enzyme activity and viral replication in cells. Further, the NS3 was found via fluorescence resonance energy transfer (FRET)-based assays to destabilize double-stranded RNA in the presence of Mg2+ or Mn2+ in an NTP-independent manner. However, the RNA destabilization activity was not affected by mutagenesis of the conserved motifs of NTPase. These results reveal that the MNV NS3 has an NTPase-independent RNA chaperone-like activity, and that a FRET-based RNA destabilization assay has the potential to identify new antiviral drugs targeting NS3.

Author Correction: Supersaturation-controlled microcrystallization and visualization analysis for serial femtosecond crystallography.

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Supersaturation-controlled microcrystallization and visualization analysis for serial femtosecond crystallography.

Time-resolved serial femtosecond crystallography with X-ray free electron laser (XFEL) holds the potential to view fast reactions occurring at near-physiological temperature. However, production and characterization of homogeneous micron-sized protein crystals at high density remain a bottleneck, due to the lack of the necessary equipments in ordinary laboratories. We describe here supersaturation-controlled microcrystallization and visualization and analysis tools that can be easily used in any laboratory. The microcrystallization conditions of the influenza virus hemagglutinin were initially obtained with low reproducibility, which was improved by employing a rapid evaporation of hanging drops. Supersaturation-controlled microcrystallization was then developed in a vapor diffusion mode, where supersaturation was induced by evaporation in hanging drops sequentially for durations ranging from 30 sec to 3 min, depending on the protein. It was applied successfully to the microcrystal formation of lysozyme, ferritin and hemagglutinin with high density. Moreover, visualization and analysis tools were developed to characterize the microcrystals observed by light microscopy. The size and density distributions of microcrystals analyzed by the tools were found to be consistent with the results of manual analysis, further validated by high-resolution microscopic analyses. Our supersaturation-controlled microcrystallization and visualization and analysis tools will provide universal access to successful XFEL studies.

Structure and Function of Caliciviral RNA Polymerases.

Caliciviruses are a leading agent of human and animal gastroenteritis and respiratory tract infections, which are growing concerns in immunocompromised individuals. However, no vaccines or therapeutics are yet available. Since the rapid rate of genetic evolution of caliciviruses is mainly due to the error-prone nature of RNA-dependent RNA polymerase (RdRp), this article focuses on recent studies of the structures and functions of RdRp from caliciviruses. It also provides recent advances in the interactions of RdRp with virion protein genome-linked (VPg) and RNA and the structural and functional features of its precursor.

Conformational modulation of influenza virus hemagglutinin: characterization and in vivo efficacy of monomeric form.

Mutational changes that mostly occur at the head region of hemagglutinin (HA) lead to the emergence of new epidemic influenza viruses, whereas HA antigens have been modified to generate broadly neutralizing antibodies toward highly conserved epitopes in the HA stem. Interestingly, a recent analysis of serum antibody repertoires showed that broadly neutralizing antibodies bind to HA monomer at a conserved region occluded at the intermonomer interface of HA trimer and confer protection in animal models. We showed previously that the recombinant HA ectodomain from a pandemic strain A/Korea/01/2009 was monomeric in solution and crystal structure. In order to examine the potential antigenicity of a monomeric form, we designed HA monomer that incorporates mutations to destabilize trimer conformations. Starting with the HA trimer from a seasonal strain A/Thailand/CU44/2006, mutations were introduced at the intermonomer interface, Ser199 of HA1 and Gly47, Arg75, Phe88, Val91, and Arg106 of HA2. Two mutants, F88E and V91W, were characterized to form a monomer and their double mutant F88E/V91W monomer was selected as an antigen. Animal studies showed that the HA monomer induced protective immunity in vivo, comparable to the trimer, albeit low antibody titers in sera.

Antiviral activities of Artemisia princeps var. orientalis essential oil and its α-thujone against norovirus surrogates.

Artemisia princeps var. orientalis is a well-known medicinal food, which has been used for the treatment of several diseases including bacterial infection. We examined the antiviral effects of the essential oil from A. princeps var. orientalis and its compounds, borneol, α-thujone and camphor, against murine norovirus-1 (MNV-1) and feline calicivirus-F9 (FCV-F9). The time-of-addition plaque assays were used to determine the ability of essential oil to interfere with viral infection. The maximum activities, following the pretreatment of FCV-F9 and MNV-1, reached 48% inhibition on FCV-F9 and 64% inhibition on MNV-1 at 0.1 and 0.01% of the essential oil, respectively. Neither borneol nor camphor exhibited an antiviral activity, whereas α-thujone, a major compound of the essential oil, showed strong inhibition on FCV-F9 and MNV-1.

Structural Basis of Novel Iron-Uptake Route and Reaction Intermediates in Ferritins from Gram-Negative Bacteria.

Iron and oxygen chemistry is mediated by iron proteins for many biological functions. Carboxylate-bridged diiron enzymes including ferritin have the common mechanism of oxygen activation via peroxodiferric intermediates. However, the route for iron uptake and the structural identification of intermediates still remain incomplete. The 4-fold symmetry channel of Helicobacter pylori ferritin was previously proposed as the iron-uptake route in eubacteria, but the amino acid residues at the 4-fold channel are not highly conserved. Here, we show evidence for a short path for iron uptake from His93 on the surface to the ferroxidase center in H. pylori ferritin and Escherichia coli ferritin. The amino acid residues along this path are highly conserved in Gram-negative bacteria and some archaea, and the mutants containing S20A and H93L showed significantly decreased iron oxidation. Surprisingly, the E. coli ferritin S20A crystal structure showed oxygen binding and side-on, symmetric µ-η2:η2 peroxodiferric and oxodiferric intermediates. The results provide the structural basis for understanding the chemical nature of intermediates in iron oxidation in bacteria and some of archaea.

Antiviral Effects of Black Raspberry (Rubus coreanus) Seed and Its Gallic Acid against Influenza Virus Infection.

Influenza is a serious public health concern worldwide, as it causes significant morbidity and mortality. The emergence of drug-resistant viral strains requires new approaches for the treatment of influenza. In this study, Rubus coreanus seed (RCS) that is left over from the production of wine or juice was found to show antiviral activities against influenza type A and B viruses. Using the time-of-addition plaque assay, viral replication was almost completely abolished by simultaneous treatment with the RCS fraction of less than a 1-kDa molecular weight (RCSF1). One of the polyphenols derived from RCSF1, gallic acid (GA), identified by liquid chromatography-tandem mass spectrometry, showed inhibitory effects against both influenza type A and B viruses, albeit at relatively high concentrations. RCSF1 was bound to hemagglutinin protein, inhibited hemagglutination significantly and disrupted viral particles, whereas GA was found to only disrupt the viral particles by using transmission electron microscopy. In BALB/c mice infected with influenza virus, oral administration of RCSF1 significantly improved the survival rate and reduced the viral titers in the lungs. Our results demonstrate that RCSF1 and GA show potent and broad antiviral activity against influenza A and B type viruses and are promising sources of agents that target virus particles.

Antiviral effects of black raspberry (Rubus coreanus) seed extract and its polyphenolic compounds on norovirus surrogates.

Black raspberry seeds, a byproduct of wine and juice production, contain large quantities of polyphenolic compounds. The antiviral effects of black raspberry seed extract (RCS) and its fraction with molecular weight less than 1 kDa (RCS-F1) were examined against food-borne viral surrogates, murine norovirus-1 (MNV-1) and feline calicivirus-F9 (FCV-F9). The maximal antiviral effect was achieved when RCS or RCS-F1 was added simultaneously to cells with MNV-1 or FCV-F9, reaching complete inhibition at 0.1-1 mg/mL. Transmission electron microscopy (TEM) images showed enlarged viral capsids or disruption (from 35 nm to up to 100 nm) by RCS-F1. Our results thus suggest that RCS-F1 can interfere with the attachment of viral surface protein to host cells. Further, two polyphenolic compounds derived from RCS-F1, cyanidin-3-glucoside (C3G) and gallic acid, identified by liquid chromatography-tandem mass spectrometry, showed inhibitory effects against the viruses. C3G was suggested to bind to MNV-1 RNA polymerase and to enlarge viral capsids using differential scanning fluorimetry and TEM, respectively.

Antiviral effects of mulberry (Morus alba) juice and its fractions on foodborne viral surrogates.

Norovirus infection is a major cause of nonbacterial foodborne outbreaks worldwide, but no specific treatments are available yet. In this study, we investigated the antiviral activity of mulberry (Morus alba, Ma) juice and its fractions on murine norovirus-1 (MNV-1) and feline calicivirus-F9 (FCV-F9) as human norovirus surrogates using cytopathic effect inhibition, plaque reduction, and RNA expression assays. In time-of-addition experiments, Ma juice was found to be effective in reducing the infectivity of MNV-1 and FCV-F9 in the pre- and co-treatments. The effective concentration for 50% reduction was approximately 0.005% juice (relative to 100% natural juice) and 0.25% juice for MNV-1 and FCV-F9, respectively. Ma juice at 0.1% exhibited about 60% reduction of the MNV-1 polymerase gene expression, confirming the inhibition of viral replication. In an attempt to identify active components with antiviral activities, Ma-F1 (<3 kDa) and Ma-F2 (>3 kDa) were examined to show that Ma-F2 was more effective than Ma-F1 in all modes, except for pre-virus treatment. Nevertheless, two major polyphenolic compounds of Ma juice, cyanidin-3-glucoside and cyanidin-3-rutinoside, showed antiviral activity in the co-treatment mode. Our results suggest that Ma juice and its fractions may inhibit internalization and replication of MNV-1, whereas it may influence adherence or internalization of FCV-F9 virions. Ma juice may prove useful in the prevention of foodborne viral infection.

Effects of Oils and Essential Oils from Seeds of Zanthoxylum schinifolium against Foodborne Viral Surrogates.

Human noroviruses are the most frequent cause of foodborne viral disease and are responsible for the vast majority of nonbacterial gastroenteritis. However, no specific therapies are available for the efficient control or prevention of foodborne viral disease. Here, we determined the antiviral activities of oils from seeds of Zanthoxylum schinifolium (ZSO) against foodborne viral surrogates, feline calicivirus-F9 (FCV-F9), and murine norovirus-1 (MNV-1), using plaque assay. Time-of-addition experiments were designed to determine the antiviral mechanism of action of ZSO against the surrogates. Maximal antiviral effect was observed upon pretreatment of FCV-F9 or MNV-1 with ZSO, which comprised oleic acid, linoleic acid, palmitic acid, and linolenic acid as the major fatty acids. FCV-F9 was more sensitive to ZSO than MNV-1, and the 50% effective concentration of ZSO against pretreatment of FCV-F9 was 0.0007%. However, essential oils from Z. schinifolium (ZSE), which comprised 42% estragole, showed no inhibitory effects against FCV-F9 and MNV-1. These results suggest that the inhibitory activities of ZSO were exerted by direct interaction of FCV-F9 or MNV-1 virion with ZSO, which may be a food material candidate for control of foodborne viral disease.

Insight into structural diversity of influenza virus haemagglutinin.

Influenza virus infects host cells through membrane fusion, a process mediated by the low pH-induced conformational change of the viral surface glycoprotein haemagglutinin (HA). We determined the structures and biochemical properties of the HA proteins from A/Korea/01/2009 (KR01), a 2009 pandemic strain, and A/Thailand/CU44/2006 (CU44), a seasonal strain. The crystal structure of KR01 HA revealed a V-shaped head-to-head arrangement, which is not seen in other HA proteins including CU44 HA. We isolated a broadly neutralizing H1-specific monoclonal antibody GC0757. The KR01 HA-Fab0757 complex structure also exhibited a head-to-head arrangement of HA. Both native and Fab complex structures reveal a different spatial orientation of HA1 relative to HA2, indicating that HA is flexible and dynamic at neutral pH. Further, the KR01 HA exhibited significantly lower protein stability and increased susceptibility to proteolytic cleavage compared with other HAs. Our structures provide important insights into the conformational flexibility of HA.