Inactivation mechanism and efficacy of grape seed extract for Human Norovirus surrogate

Proper disinfection of harvested food and water is critical to minimize infectious disease. Grape seed extract (GSE), a commonly used health supplement, is a mixture of plant-derived polyphenols. Polyphenols possess anti-microbial and -fungal properties, but anti-viral effects are not well-known. Here we show that GSE outperformed chemical disinfectants (e.g., free chlorine and peracetic acids) in inactivating Tulane virus, a human norovirus surrogate. GSE induced virus aggregation, an event that correlated with a decrease in virus titers. This aggregation and disinfection was not reversible. Molecular docking simulations indicate that polyphenols potentially formed hydrogen bonds and strong hydrophobic interactions with specific residues in viral capsid proteins. Together, these data suggest that polyphenols physically associate with viral capsid proteins to aggregate viruses as a means to inhibit virus entry into the host cell. Plant-based polyphenols like GSE are an attractive alternative to chemical disinfectants to remove infectious viruses from water or food.ImportanceHuman noroviruses are major food- and water-borne pathogens, causing approximately 20% of all cases of acute gastroenteritis cases in developing and developed countries. Proper sanitation or disinfection are critical strategies to minimize human norovirus-caused disease until a reliable vaccine is created. Grape seed extract (GSE) is a mixture of plant-derived polyphenols that is used as a health supplement. Polyphenols are known for antimicrobial, antifungal, and antibiofilm activities, but antiviral effects are not well-known. In studies here, plant-derived polyphenols outperformed chemical disinfectants (e.g., free chlorine and peracetic acids) in inactivating Tulane virus, a human norovirus surrogate. Based on data from additional molecular assays and molecular docking simulations, the current model is that the polyphenols in GSE bind to the Tulane virus capsid, an event that triggers virion aggregation. It is thought that this aggregation prevents Tulane virus from entering host cells.

A Small Virus to Deliver Small Antibodies: New Targeted Therapies Based on AAV Delivery of Nanobodies

Nanobodies are camelid-derived single-domain antibodies that present some advantages versus conventional antibodies, such as a smaller size, and higher tissue penetrability, stability, and hydrophilicity. Although nanobodies can be delivered as proteins, in vivo expression from adeno-associated viral (AAV) vectors represents an attractive strategy. This is due to the fact that AAV vectors, that can provide long-term expression of recombinant genes, have shown an excellent safety profile, and can accommodate genes for one or several nanobodies. In fact, several studies showed that AAV vectors can provide sustained nanobody expression both locally or systemically in preclinical models of human diseases. Some of the pathologies addressed with this technology include cancer, neurological, cardiovascular, infectious, and genetic diseases. Depending on the indication, AAV-delivered nanobodies can be expressed extracellularly or inside cells. Intracellular nanobodies or “intrabodies” carry out their function by interacting with cell proteins involved in disease and have also been designed to help elucidate cellular mechanisms by interfering with normal cell processes. Finally, nanobodies can also be used to retarget AAV vectors, when tethered to viral capsid proteins. This review covers applications in which AAV vectors have been used to deliver nanobodies, with a focus on their therapeutic use.

Biosynthesis of linear protein nanoarrays using the flagellar axoneme

Applications in biotechnology and synthetic biology often make use of soluble proteins, but there are many potential advantages to anchoring enzymes to a stable substrate, including stability and the possibility for substrate channeling. To avoid the necessity of protein purification and chemical immobilization, there has been growing interest in bio-assembly of protein-containing nanoparticles, exploiting the self-assembly of viral capsid proteins or other proteins that form polyhedral structures. But these nanoparticle are limited in size which constrains the packaging and the accessibility of the proteins. The axoneme, the insoluble protein core of the eukaryotic flagellum or cilium, is a highly ordered protein structure that can be several microns in length, orders of magnitude larger than other types of nanoparticles. We show that when proteins of interest are fused to specific axonemal proteins and expressed in living cells, they become incorporated into linear arrays which have the advantages of high protein loading capacity, high stability, and single-step purification with retention of biomass. The arrays can be isolated as membrane enclosed vesicle or as exposed protein arrays. The approach is demonstrated for both fluorescent proteins and enzymes, and in the latter case it is found that incorporation into axoneme arrays provides increased stability for the enzyme.

Co-expression of double-stranded RNA and viral capsid protein in the novel engineered Escherichia coli DualX-B15(DE3) strain

Background Viruses cause significant economic losses to shrimp aquaculture worldwide. In severe cases, they can lead to 100% mortality within a matter of days, hence the aquaculture industry requires antiviral strategies to minimize economic impacts. Currently, a double-stranded RNA (dsRNA)-based platform has been proven effective at a laboratory scale. The bottleneck for its industrialization is the lack of low-cost, efficient and practical delivery approaches. In an effort to bridge the gap between laboratory and farm applications, virus-like particles (VLP) have been used as nanocarriers of dsRNA. However, the implementation of this approach still suffers from high costs and a lengthy procedure, co-expression of subunits of VLP or capsid proteins (CPs) and dsRNA can be the solution for the problem. CP and dsRNA are traditionally expressed in two different E. coli hosts: protease-deficient and RNase III-deficient strains. To condense the manufacturing of dsRNA-containing VLP, this study constructed a novel E. coli strain that is able to co-express viral capsid proteins and dsRNA in the same E. coli cell. Results A novel bacterial strain DualX-B15(DE3) was engineered to be both protease- and RNase III-deficiency via P1 phage transduction. The results revealed that it could simultaneously express recombinant proteins and dsRNA. Conclusion Co-expression of viral capsid proteins and dsRNA in the same cell has been shown to be feasible. Not only could this platform serve as a basis for future cost-effective and streamlined production of shrimp antiviral therapeutics, it may be applicable for other applications that requires co-expression of recombinant proteins and dsRNA.

Adenovirus Core Proteins: Structure and Function

Adenoviruses have served as a model for investigating viral-cell interactions and discovering different cellular processes, such as RNA splicing and DNA replication. In addition, the development and evaluation of adenoviruses as the viral vectors for vaccination and gene therapy has led to detailed investigations about adenovirus biology, including the structure and function of the adenovirus encoded proteins. While the determination of the structure and function of the viral capsid proteins in adenovirus biology has been the subject of numerous reports, the last few years have seen increased interest in elucidating the structure and function of the adenovirus core proteins. Here, we provide a review of research about the structure and function of the adenovirus core proteins in adenovirus biology.

Specificity Determination in Saccharomyces cerevisiae Killer Virus Systems

Saccharomyces yeasts are widely distributed in the environment and microbiota of higher organisms. The killer phenotype of yeast, encoded by double-stranded RNA (dsRNA) virus systems, is a valuable trait for host survival. The mutual relationship between the different yet clearly defined LA and M virus pairs suggests complex fitting context. To define the basis of this compatibility, we established a system devoted to challenging inherent yeast viruses using viral proteins expressed in trans. Virus exclusion by abridged capsid proteins was found to be complete and nonspecific, indicating the presence of generic mechanisms of Totiviridae maintenance in yeast cells. Indications of specificity in both the exclusion of LA viruses and the maintenance of M viruses by viral capsid proteins expressed in trans were observed. This precise specificity was further established by demonstrating the importance of the satellite virus in the maintenance of LA virus, suggesting the selfish behavior of M dsRNA.

Advantages and Prospects of Tag/Catcher Mediated Antigen Display on Capsid-Like Particle-Based Vaccines

Capsid-like particles (CLPs) are multimeric, repetitive assemblies of recombinant viral capsid proteins, which are highly immunogenic due to their structural similarity to wild-type viruses. CLPs can be used as molecular scaffolds to enable the presentation of soluble vaccine antigens in a similar structural format, which can significantly increase the immunogenicity of the antigen. CLP-based antigen display can be obtained by various genetic and modular conjugation methods. However, these vary in their versatility as well as efficiency in achieving an immunogenic antigen display. Here, we make a comparative review of the major CLP-based antigen display technologies. The Tag/Catcher-AP205 platform is highlighted as a particularly versatile and efficient technology that offers new qualitative and practical advantages in designing modular CLP vaccines. Finally, we discuss how split-protein Tag/Catcher conjugation systems can help to further propagate and enhance modular CLP vaccine designs.

Discovery of several thousand highly diverse circular DNA viruses

Although millions of distinct virus species likely exist, only approximately 9000 are catalogued in GenBank's RefSeq database. We selectively enriched for the genomes of circular DNA viruses in over 70 animal samples, ranging from nematodes to human tissue specimens. A bioinformatics pipeline, Cenote-Taker, was developed to automatically annotate over 2500 complete genomes in a GenBank-compliant format. The new genomes belong to dozens of established and emerging viral families. Some appear to be the result of previously undescribed recombination events between ssDNA and ssRNA viruses. In addition, hundreds of circular DNA elements that do not encode any discernable similarities to previously characterized sequences were identified. To characterize these ‘dark matter’ sequences, we used an artificial neural network to identify candidate viral capsid proteins, several of which formed virus-like particles when expressed in culture. These data further the understanding of viral sequence diversity and allow for high throughput documentation of the virosphere.