Chromatographic Purification and Polishing of AAV Particles.

The production of Adeno-associated virus (AAV) vectors in the lab setting has typically involved expression in adherent cells followed by purification through ultracentrifugation in density gradients. This production method is, however, not easily scalable, presents high levels of cellular impurities that co-purify with the virus, and results in a mixture of empty and full capsids. Here we describe a detailed AAV production protocol that overcomes these limitations through AAV expression in suspension cells followed by AAV affinity purification and AAV polishing to separate empty and full capsids, resulting in high yields of ultra-pure AAV that is highly enriched in full capsids.

Stable coexistence between an archaeal virus and the dominant methanogen of the human gut.

The human gut virome, which is mainly composed of bacteriophages, also includes viruses infecting archaea, yet their role remains poorly understood due to lack of isolates. Here, we characterize a temperate archaeal virus (MSTV1) infecting Methanobrevibacter smithii, the dominant methanogenic archaeon of the human gut. The MSTV1 genome is integrated in the host chromosome as a provirus which is sporadically induced, resulting in virion release. Using cryo-electron tomography, we capture several intracellular virion assembly intermediates and confirm that only a small fraction of the host population actively produces virions in vitro. Similar low frequency of induction is observed in a mouse colonization model, using mice harboring a stable consortium of 12 bacterial species (OMM12). Transcriptomic analysis suggests a regulatory lysogeny-lysis switch involving an interplay between viral proteins to maintain virus-host equilibrium, ensuring host survival and viral persistence. Thus, our study sheds light on archaeal virus-host interactions and highlights similarities with bacteriophages in establishing stable coexistence with their hosts in the gut.

HIV-1 adapts to lost IP6 coordination through second-site mutations that restore conical capsid assembly.

The HIV-1 capsid is composed of capsid (CA) protein hexamers and pentamers (capsomers) that contain a central pore hypothesised to regulate capsid assembly and facilitate nucleotide import early during post-infection. These pore functions are mediated by two positively charged rings created by CA Arg-18 (R18) and Lys-25 (K25). Here we describe the forced evolution of viruses containing mutations in R18 and K25. Whilst R18 mutants fail to replicate, K25A viruses acquire compensating mutations that restore nearly wild-type replication fitness. These compensating mutations, which rescue reverse transcription and infection without reintroducing lost pore charges, map to three adaptation hot-spots located within and between capsomers. The second-site suppressor mutations act by restoring the formation of pentamers lost upon K25 mutation, enabling closed conical capsid assembly both in vitro and inside virions. These results indicate that there is no intrinsic requirement for K25 in either nucleotide import or capsid assembly. We propose that whilst HIV-1 must maintain a precise hexamer:pentamer equilibrium for proper capsid assembly, compensatory mutations can tune this equilibrium to restore fitness lost by mutation of the central pore.

The HIV-1 capsid serves as a nanoscale reaction vessel for reverse transcription.

The viral capsid performs critical functions during HIV-1 infection and is a validated target for antiviral therapy. Previous studies have established that the proper structure and stability of the capsid are required for efficient HIV-1 reverse transcription in target cells. Moreover, it has recently been demonstrated that permeabilized virions and purified HIV-1 cores undergo efficient reverse transcription in vitro when the capsid is stabilized by addition of the host cell metabolite inositol hexakisphosphate (IP6). However, the molecular mechanism by which the capsid promotes reverse transcription is undefined. Here we show that wild type HIV-1 virions can undergo efficient reverse transcription in vitro in the absence of a membrane-permeabilizing agent. This activity, originally termed "natural endogenous reverse transcription" (NERT), depends on expression of the viral envelope glycoprotein during virus assembly and its incorporation into virions. Truncation of the gp41 cytoplasmic tail markedly reduced NERT activity, suggesting that gp41 licenses the entry of nucleotides into virions. By contrast to reverse transcription in permeabilized virions, NERT required neither the addition of IP6 nor a mature capsid, indicating that an intact viral membrane can substitute for the function of the viral capsid during reverse transcription in vitro. Collectively, these results demonstrate that the viral capsid functions as a nanoscale container for reverse transcription during HIV-1 infection.

Inherent symmetry and flexibility in hepatitis B virus subviral particles.

Chronic hepatitis B virus (HBV) infection poses a major global health challenge with massive morbidity and mortality. Despite a preventive vaccine, current treatments provide limited virus clearance, necessitating lifelong commitment. The HBV surface antigen (HBsAg) is crucial for diagnosis and prognosis, yet its high-resolution structure and assembly on the virus envelope remain elusive. Utilizing extensive datasets and advanced cryo-electron microscopy analysis, we present structural insights into HBsAg at a near-atomic resolution of 3.7 angstroms. HBsAg homodimers assemble into subviral particles with D2- and D4-like quasisymmetry, elucidating the dense-packing rules and structural adaptability of HBsAg. These findings provide insights into how HBsAg assembles into higher-order filaments and interacts with the capsid to form virions.

Delivery of US28 by incoming HCMV particles rapidly attenuates Akt activity to suppress HCMV lytic replication in monocytes.

Establishing a nonproductive, quiescent infection within monocytes is essential for the spread of human cytomegalovirus (HCMV). We investigated the mechanisms through which HCMV establishes a quiescent infection in monocytes. US28 is a virally encoded G protein-coupled receptor (GPCR) that is essential for silent infections within cells of the myeloid lineage. We found that preformed US28 was rapidly delivered to monocytes by HCMV viral particles, whereas the de novo synthesis of US28 was delayed for several days. A recombinant mutant virus lacking US28 (US28Δ) was unable to establish a quiescent infection, resulting in a fully productive lytic infection able to produce progeny virus. Infection with US28Δ HCMV resulted in the phosphorylation of the serine and threonine kinase Akt at Ser473 and Thr308, in contrast with the phosphorylation of Akt only at Ser473 after WT viral infection. Inhibiting the dual phosphorylation of Akt prevented the lytic replication of US28Δ, and ectopic expression of a constitutively phosphorylated Akt variant triggered lytic replication of wild-type HCMV. Mechanistically, we found that US28 was necessary and sufficient to attenuate epidermal growth factor receptor (EGFR) signaling induced during the entry of WT virus, which led to the site-specific phosphorylation of Akt at Ser473. Thus, particle-delivered US28 fine-tunes Akt activity by limiting HCMV-induced EGFR activation during viral entry, enabling quiescent infection in monocytes.

Insights on the Mechanical Properties of SARS-CoV-2 Particles and the Effects of the Photosensitizer Hypericin.

SARS-CoV-2 is a highly pathogenic virus responsible for the COVID-19 disease. It belongs to the Coronaviridae family, characterized by a phospholipid envelope, which is crucial for viral entry and replication in host cells. Hypericin, a lipophilic, naturally occurring photosensitizer, was reported to effectively inactivate enveloped viruses, including SARS-CoV-2, upon light irradiation. In addition to its photodynamic activity, Hyp was found to exert an antiviral action also in the dark. This study explores the mechanical properties of heat-inactivated SARS-CoV-2 viral particles using Atomic Force Microscopy (AFM). Results reveal a flexible structure under external stress, potentially contributing to the virus pathogenicity. Although the fixation protocol causes damage to some particles, correlation with fluorescence demonstrates colocalization of partially degraded virions with their genome. The impact of hypericin on the mechanical properties of the virus was assessed and found particularly relevant in dark conditions. These preliminary results suggest that hypericin can affect the mechanical properties of the viral envelope, an effect that warrants further investigation in the context of antiviral therapies.

A Novel Application of Virus Like Particles in the Hemagglutination Inhibition Assay.

The hemagglutination inhibition (HI) assay is a traditional laboratory procedure for detection and quantitation of serum antibodies of hemagglutinating viruses containing the hemagglutinin (HA) gene. The current study aimed to investigate the novel use of virus like particles (VLP) as an antigen for the HI assay. VLPs were prepared from a strain of H5N1 using a baculovirus expression system. The VLPs were characterized using the hemagglutination test, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and transmission electron microscopy. The comparative HI assay was performed using three different seed antigens: A/chicken/Mexico/232/94 (H5N2), A/chicken/Egypt/18-H/09(H5N1) and A/goose/Guangdong/1/1996(H5N1). The HI assay of serum antibody titrations using homologous antigens to these vaccinal seeds were compared to the VLP's antigens for the same serum. The HI titers were logically relevant to the similarity between VLP antigens and vaccinal seeds, indicating the VLPs behave similarly to the standard HI assay which uses inactivated whole virus as an antigen. VLPs could be considered as an alternative to the HI assay antigen as they show a relatedness between the similarity with vaccinal seed and serum antibodies. Compared to typical entire H5N1 viral antigen prepared in SPF eggs that require proper inactivation to avoid any public health risk, VLPs prepared in tissue culture, plants or insect cells are a safe, inexpensive and scalable alternative to inactivated whole virus antigen.

Shedding Light on Viral Shedding: Novel Insights into Nuclear Assembly, Cytoplasmic Transformation and Extracellular Vesicle Release of the BK Virus.

Despite the high prevalence of BK polyomavirus (BKPyV) and the associated risk for BKPyV-associated nephropathy (BKPyVAN) in kidney transplant (KTX) recipients, many details on viral processes such as replication, maturation, assembly and virion release from host cells have not been fully elucidated. VP1 is a polyomavirus-specific protein that is expressed in the late phase of its replicative cycle with important functions in virion assembly and infectious particle release. This study investigated the localization and time-dependent changes in the distribution of VP1-positive viral particles and their association within the spectrum of differing cell morphologies that are observed in the urine of KTX patients upon active BKPyV infection. We found highly differing recognition patterns of two anti-VP1 antibodies with respect to intracellular and extracellular VP1 localization, pointing towards independent binding sites that were seemingly associated with differing stages of virion maturation. Cells originating from single clones were stably cultured out of the urine sediment of KTX recipients with suspected BKPyVAN. The cell morphology, polyploidy, virus replication and protein production were investigated by confocal microscopy using both a monoclonal (mAb 4942) and a polyclonal rabbit anti-VP1-specific antibody (RantiVP1 Ab). Immunoblotting was performed to investigate changes in the VP1 protein. Both antibodies visualized VP1 and the mAb 4942 recognized VP1 in cytoplasmic vesicles exhibiting idiomorphic sizes when released from the cells. In contrast, the polyclonal antibody detected VP1 within the nucleus and in cytoplasm in colocalization with the endoplasmic reticulum marker CNX. At the nuclear rim, VP1 was recognized by both antibodies. Immunoblotting revealed two smaller versions of VP1 in urinary decoy cell extracts, potentially from different translation start sites as evaluated by in silico analysis. Oxford Nanopore sequencing showed integration of BKPyV DNA in chromosomes 3, 4 and 7 in one of the five tested primary cell lines which produced high viral copies throughout four passages before transcending into senescence. The different staining with two VP1-specific antibodies emphasizes the modification of VP1 during the process of virus maturation and cellular exit. The integration of BKPyV into the human genome leads to high virus production; however, this alone does not transform the cell line into a permanently cycling and indefinitely replicating one.

Purification of Epizootic Hemorrhagic Disease Virus (and Other Orbiviruses) Particles from Infected Mammalian or Insect Cells.

Epizootic hemorrhagic disease virus (EHDV), like other orbiviruses, infects and replicates in mammalian and insect vector cells. Within its ruminant hosts EHDV, like bluetongue virus (BTV), it has mainly been associated with infection of endothelial cells of capillaries as well as leukocyte subsets. Furthermore, EHDV infects and replicates within its biological vector, Culicoides biting midges and Culicoides-derived cells. A wide range of common laboratory cell lines such as BHK, BSR, and Vero cells are susceptible to infection with certain EHDV strains. Cell culture supernatants of infected cells are commonly used for both in vivo and in vitro infection studies. For specific virological or immunological studies, using highly purified virus particles, however, might be beneficial or even required. Here we describe a purification method for EHDV particles, which had been originally developed for certain strains of BTV.

Chemically Tagging Cargo for Specific Packaging inside and on the Surface of Virus-like Particles.

Virus-like particles (VLPs) have untapped potential for packaging and delivery of macromolecular cargo. To be a broadly useful platform, there needs to be a strategy for attaching macromolecules to the inside or the outside of the VLP with minimal modification of the platform or cargo. Here, we repurpose antiviral compounds that bind to hepatitis B virus (HBV) capsids to create a chemical tag to noncovalently attach cargo to the VLP. Our tag consists of a capsid assembly modulator, HAP13, connected to a linker terminating in maleimide. Our cargo is a green fluorescent protein (GFP) with a single addressable cysteine, a feature that can be engineered in many proteins. The HAP-GFP construct maintained HAP's intrinsic ability to bind HBV capsids and accelerate assembly. We investigated the capacity of HAP-GFP to coassemble with HBV capsid protein and bind to preassembled capsids. HAP-GFP binding was concentration-dependent, sensitive to capsid stability, and dependent on linker length. Long linkers had the greatest activity to bind capsids, while short linkers impeded assembly and damaged intact capsids. In coassembly reactions, >20 HAP-GFP molecules were presented on the outside and inside of the capsid, concentrating the cargo by more than 100-fold compared to bulk solution. We also tested an HAP-GFP with a cleavable linker so that external GFP molecules could be removed, resulting in exclusive internal packaging. These results demonstrate a generalizable strategy for attaching cargo to a VLP, supporting development of HBV as a modular VLP platform.

Role of Protein VII in the Production of Infectious Bovine Adenovirus-3 Virion.

Bovine adenovirus (BAdV)-3 genome encodes a 26 kDa core protein designated as protein VII, which localizes to the nucleus/nucleolus. The requirement of a protein VII-complementing cell line for the replication of VII-deleted BAdV-3 suggests that protein VII is required for the production of infectious progeny virions. An analysis of the BAV.VIId+ virus (only phenotypically positive for protein VII) detected no noticeable differences in the expression and incorporation of viral proteins in the virions. Moreover, protein VII does not appear to be essential for the formation of mature BAV.VIId+. However, protein VII appeared to be required for the efficient assembly of mature BAV.VIId- virions. An analysis of the BAV.VIId- virus (genotypically and phenotypically negative for protein VII) in non-complementing cells detected the inefficient release of virions from endosomes, which affected the expression of viral proteins or DNA replication. Moreover, the absence of protein VII altered the proteolytic cleavage of protein VI of BAV.VIId-. Our results suggest that BAdV-3 protein VII appears to be required for efficient production of mature virions. Moreover, the absence of protein VII produces non-infectious BAdV-3 by altering the release of BAdV-3 from endosomes/vesicles.

Baculovirus expression and purification of virion core and envelope proteins of goatpox virus to evaluate their diagnostic potential.

Goatpox and sheeppox are highly contagious and economically important viral diseases of small ruminants. Due to the risk they pose to animal health, livestock production, and international trade, capripoxviruses are a considerable threat to the livestock economy. In this study, we expressed two core proteins (A4L and A12L) and one extracellular enveloped virion protein (A33R) of goatpox virus in a baculovirus expression vector system and evaluated their use as diagnostic antigens in ELISA. Full-length A4L, A12L, and A33R genes of the GTPV Uttarkashi strain were amplified, cloned into the pFastBac HT A donor vector, and introduced into DH10Bac cells containing a baculovirus shuttle vector plasmid to generate recombinant bacmids. The recombinant baculoviruses were produced in Sf-21 cells by transfection, and proteins were expressed in TN5 insect cells. The recombinant proteins were analysed by SDS-PAGE and confirmed by western blot, with expected sizes of ~30 kDa, ~31 kDa, and ~32 kDa for A4L, A12L, and A33R, respectively. The recombinant proteins were purified, and the immunoreactivity of the purified proteins was confirmed by western blot using anti-GTPV serum. The antigenic specificity of the expressed proteins as diagnostic antigens was evaluated by testing their reactivity with infected, vaccinated, and negative GTPV/SPPV serum in indirect ELISA, and the A33R-based indirect ELISA was optimized. The diagnostic sensitivity and specificity of the A33R-based indirect ELISA were found to be of 89% and 94% for goats and 98% and 91%, for sheep, respectively. No cross-reactivity was observed with other related viruses. The recombinant-A33R-based indirect ELISA developed in the present study shows that it has potential for the detection of antibodies in GTPV and SPPV infected/vaccinated animals.

Structure of anellovirus-like particles reveal a mechanism for immune evasion.

Anelloviruses are nonpathogenic viruses that comprise a major portion of the human virome. Despite being ubiquitous in the human population, anelloviruses (ANVs) remain poorly understood. Basic features of the virus, such as the identity of its capsid protein and the structure of the viral particle, have been unclear until now. Here, we use cryogenic electron microscopy to describe the first structure of an ANV-like particle. The particle, formed by 60 jelly roll domain-containing ANV capsid proteins, forms an icosahedral particle core from which spike domains extend to form a salient part of the particle surface. The spike domains come together around the 5-fold symmetry axis to form crown-like features. The base of the spike domain, the P1 subdomain, shares some sequence conservation between ANV strains while a hypervariable region, forming the P2 subdomain, is at the spike domain apex. We propose that this structure renders the particle less susceptible to antibody neutralization by hiding vulnerable conserved domains while exposing highly diverse epitopes as immunological decoys, thereby contributing to the immune evasion properties of anelloviruses. These results shed light on the structure of anelloviruses and provide a framework to understand their interactions with the immune system.

Nectin1 is a pivotal host factor involved in attachment and entry of red-spotted grouper nervous necrosis virus in the early stages of the viral life cycle.

Nervous necrosis virus (NNV) is a highly neurotropic virus that poses a persistent threat to the survival of multiple fish species. However, its inimitable neuropathogenesis remains largely elusive. To rummage potential partners germane to the nervous system, we investigated the interaction between red-spotted grouper NNV (RGNNV) and grouper brain by immunoprecipitation coupled with mass spectrometry and discerned Nectin1 as a novel host factor subtly involved in viral early invasion events. Nectin1 was abundant in neural tissues and implicated in the inception of tunnel nanotubes triggered by RGNNV. Its overexpression not only dramatically potentiated the replication dynamics of RGNNV in susceptible cells, but also empowered non-sensitive cells to expeditiously capture free virions within 2 min. This potency was impervious to low temperatures but was dose-dependently suppressed by soluble protein or specific antibody of Nectin1 ectodomain, indicating Nectin1 as an attachment receptor for RGNNV. Mechanistically, efficient hijacking of virions by Nectin1 strictly depended on intricate linkages to different modules of viral capsid protein, especially the direct binding between the IgC1 loop and P-domain. More strikingly, despite abortive proliferation in Nectin1-reconstructed CHSE-214 cells, a non-sensitive cell, RGNNV could gain access to the intracellular compartment by capitalizing on Nectin1, thereby inducing canonical cytoplasmic vacuolation. Altogether, our findings delineate a candidate entrance for RGNNV infiltration into the nervous system, which may shed unprecedented insights into the exploration and elucidation of RGNNV pathogenesis.IMPORTANCENervous necrosis virus (NNV) is one of the most virulent pathogens in the aquaculture industry, which inflicts catastrophic damage to ecology, environment, and economy annually around the world. Nevertheless, its idiosyncratic invasion and latency mechanisms pose enormous hardships to epidemic prevention and control. In this study, deploying grouper brain as a natural screening library, a single-transmembrane glycoprotein, Nectin1, was first identified as an emergent functional receptor for red-spotted grouper NNV (RGNNV) that widely allocated in nervous tissues and directly interacted with viral capsid protein through distinct Ig-like loops to bridge virus-host crosstalk, apprehend free virions, and concomitantly propel viral entry. Our findings illuminate the critical role of Nectin1 in RGNNV attachment and entry and provide a potential target for future clinical intervention strategies in the therapeutic race against RGNNV.

Subnanometer structure of medusavirus capsid during maturation using cryo-electron microscopy.

Medusavirus is a giant virus classified into an independent family of Mamonoviridae. Amoebae infected with medusavirus release immature particles in addition to virions. These particles were suggested to exhibit the maturation process of this virus, but the structure of these capsids during maturation remains unknown. Here, we apply a block-based reconstruction method in cryo-electron microscopy (cryo-EM) single particle analysis to these viral capsids, extending the resolution to 7-10 Å. The maps reveal a novel network composed of minor capsid proteins (mCPs) supporting major capsid proteins (MCPs). A predicted molecular model of the MCP fitted into the cryo-EM maps clarified the boundaries between the MCP and the underlining mCPs, as well as between the MCP and the outer spikes, and identified molecular interactions between the MCP and these components. Several structural changes of the mCPs under the fivefold vertices of the immature particles were observed, depending on the presence or absence of the underlying internal membrane. In addition, the lower part of the penton proteins on the fivefold vertices was also missing in mature virions. These dynamic conformational changes of mCPs indicate an important function in the maturation process of medusavirus.IMPORTANCEThe structural changes of giant virus capsids during maturation have not thus far been well clarified. Medusavirus is a unique giant virus in which infected amoebae release immature particles in addition to mature virus particles. In this study, we used cryo-electron microscopy to investigate immature and mature medusavirus particles and elucidate the structural changes of the viral capsid during the maturation process. In DNA-empty particles, the conformation of the minor capsid proteins changed dynamically depending on the presence or absence of the underlying internal membranes. In DNA-full particles, the lower part of the penton proteins was lost. This is the first report of structural changes of the viral capsid during the maturation process of giant viruses.

Gene Editing by Ferrying of CRISPR/Cas Ribonucleoprotein Complexes in Enveloped Virus-Derived Particles.

The invention of next-generation CRISPR/Cas gene editing tools, like base and prime editing, for correction of gene variants causing disease, has created hope for in vivo use in patients leading to wider clinical translation. To realize this potential, delivery vehicles that can ferry gene editing tool kits safely and effectively into specific cell populations or tissues are in great demand. In this review, we describe the development of enveloped retrovirus-derived particles as carriers of "ready-to-work" ribonucleoprotein complexes consisting of Cas9-derived editor proteins and single guide RNAs. We present arguments for adapting viruses for cell-targeted protein delivery and describe the status after a decade-long development period, which has already shown effective editing in primary cells, including T cells and hematopoietic stem cells, and in tissues targeted in vivo, including mouse retina, liver, and brain. Emerging evidence has demonstrated that engineered virus-derived nanoparticles can accommodate both base and prime editors and seems to fertilize a sprouting hope that such particles can be further developed and produced in large scale for therapeutic applications.

Mouse hepatitis virus JHMV I protein is required for maximal virulence.

Betacoronaviruses encode a conserved accessory gene within the +1 open reading frame (ORF) of nucleocapsid called the internal N gene. This gene is referred to as "I" for mouse hepatitis virus (MHV), ORF9b for severe acute respiratory CoV (SARS-CoV) and SARS-CoV-2, and ORF8b for Middle East respiratory syndrome CoV (MERS-CoV). Previous studies have shown ORF8b and ORF9b have immunoevasive properties, while the only known information for MHV I is its localization within the virion of the hepatotropic/neurotropic A59 strain of MHV. Whether MHV I is an innate immune antagonist or has other functions has not been evaluated. In this report, we show that the I protein of the neurotropic JHM strain of MHV (JHMV) lacks a N terminal domain present in other MHV strains, has immunoevasive properties, and is a component of the virion. Genetic deletion of JHMV I (rJHMVIΔ57-137) resulted in a highly attenuated virus both in vitro and in vivo that displayed a post RNA replication/transcription defect that ultimately resulted in fewer infectious virions packaged compared with wild-type virus. This phenotype was only seen for rJHMVIΔ57-137, suggesting the structural changes predicted for A59 I altered its function, as genetic deletion of A59 I did not change viral replication or pathogenicity. Together, these data show that JHMV I both acts as a mild innate immune antagonist and aids in viral assembly and infectious virus production, and suggest that the internal N proteins from different betacoronaviruses have both common and virus strain-specific properties.IMPORTANCECoV accessory genes are largely studied in overexpression assays and have been identified as innate immune antagonists. However, functions identified after overexpression are often not confirmed in the infected animal host. Furthermore, some accessory proteins are components of the CoV virion, but their role in viral replication and release remains unclear. Here, we utilized reverse genetics to abrogate expression of a conserved CoV accessory gene, the internal N ("I") gene, of the neurotropic JHMV strain of MHV and found that loss of the I gene resulted in a post replication defect that reduced virion assembly and ultimately infectious virus production, while also increasing some inflammatory molecule expression. Thus, the JHMV I protein has roles in virion assembly that were previously underappreciated and in immunoevasion.

Aptamer-quantum dots platform for SARS-CoV-2 viral particle detection by fluorescence microscopy.

The virus is the smallest known replicative unit, usually in nanometer-range sizes. The most simple and sensitive detection assay involves molecular amplification of nucleic acids. This work shows a novel, straightforward detection based on the interaction of viral particles with fluorescent nanoconstructs without using enzymatic amplification, washing or separation steps. Fluorescent nanoconstructs are prepared with individual quantum dots of different emitting green and red fluorescence as a core. They are decorated with aptamers developed to recognise the receptor-binding region of the SARS-CoV-2 spike protein. Nanoconstructs can recognise SARS-CoV-2 viral particles fixed onto a coverglass generating aggregates. Meanwhile, SARS-CoV-2 viral particles/nanoconstructs complexes in solution yield aggregates and complexes, which a fluorescence microscope can visualise. The multiple molecular recognition allowed the detection of SARS-CoV-2 viral particles from a few microliters of patient swabs. This specific SARS-CoV-2/nanoconstructs interaction generates insoluble and precipitating aggregates. By using a mixture of green and red fluorescent nanoconstructs, upon the viral particle interaction, they yield heterochromatic green, red and yellow spectral fluorescence, easily identifiable by a fluorescence microscope. Washing and separation steps are not required, and aggregates allow one to easily recognise them, offering a sensitive, simple, and cheap alternative for viral detection.

Inhibition of Norovirus GII.4 binding to HBGAs by Sargassum fusiforme polysaccharide.

Norovirus (NoV) is the main pathogen that causes acute gastroenteritis and brings a heavy socio-economic burden worldwide. In this study, five polysaccharide fractions, labeled pSFP-1-5, were isolated and purified from Sargassum fusiforme (S. fusiforme). In vitro experiments demonstrated that pSFP-5 significantly prevented the binding of type A, B and H histo-blood group antigens (HBGAs) to NoV GII.4 virus-like particles (NoV GII.4 VLPs). In addition, in vivo experiments revealed that pSFP-5 was effective in reducing the accumulation of NoV in oysters, indicating that pSFP-5 could reduce the risk of NoV infection from oyster consumption. The results of transmission electron microscopy showed that the appearance of NoV GII.4 VLPs changed after pSFP-5 treatment, indicating that pSFP-5 may achieve antiviral ability by altering the morphological structure of the viral particles so that they could not bind to HBGAs. The results of the present study indicate that pSFP-5 may be an effective anti-NoV substance and can be used as a potential anti-NoV drug component.