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Introduction: Enteroviruses are common viruses causing a huge number of acute and chronic infections and producing towering economic costs. Similarly, coronaviruses cause seasonal mild infections, epidemics, and even pandemics and can lead to severe respiratory symptoms. It is important to develop broadly acting antiviral molecules to efficiently tackle the infections caused by thes. Areas covered: This review illuminates the differences and similarities between enteroviruses and coronaviruses and examines the most appealing therapeutic targets to combat both virus groups. Publications of both virus groups and deposited structures discovered through PubMed to March 2021 for viral proteases have been evaluated. Expert opinion: The main protease of coronaviruses and enteroviruses share similarities in their structure and function. These proteases process their viral polyproteins and thus drugs that bind to the active site have potential to target both virus groups. It is important to develop drugs that target more evolutionarily conserved processes and proteins. Moreover, it is a wise strategy to concentrate on processes that are similar between several virus families.

Interactions between an atomically precise gold nanocluster Au102(p-MBA)44 (p-MBA = para mercaptobenzoic acid) and a fluorescent organic dye molecule (KU, azadioxatriangulenium) are studied. In solution, the constituents form spontaneously a weakly bound complex leading to quenching of fluorescence of the KU dye via energy transfer. The KU can be separated from the complex by lowering pH, leading to recovery of fluorescence, which forms a basis for an optical reversible pH sensor. However, the sensor is not a stable entity, which could be delivered inside cells. For this purpose, a covalently bound hybrid is synthesized by linking the KU dye to the ligand layer of the cluster via an ester bond. Covalent linking facilitates entry of the cluster–dye hybrids into cells via endocytosis. Inside cells, the hybrids accumulate in endosomes where Au102 releases its cargo via hydrolysis of the ester bond. Changes of the local pH inside endosomes regulate reversible fluorescence due to variations in the interactions between the Au102 cluster and the dye. This work presents a concept for delivering reporter molecules into cells by using atomically precise gold nanoclusters as carriers and paves the way for future developments of cluster-reporter sensors for in vivo measurements of e.g. absolute pH values or ion concentrations.

The worldwide spread of the SARS-CoV-2 virus has continued to accelerate, putting a considerable burden on public health, safety, and the global economy. Taking into consideration that the main route of virus transmission is via respiratory particles, the face mask represents a simple and efficient barrier between potentially infected and healthy individuals, thus reducing transmissibility per contact by reducing transmission of infected respiratory particles. However, long-term usage of a face mask leads to the accumulation of significant amounts of different pathogens and viruses onto the surface of the mask and can result in dangerous bacterial and viral co-infections. Zeolite imidazolate framework-8 (ZIF-8) has recently emerged as an efficient water-stable photocatalyst capable of generating reactive oxygen species under light irradiation destroying dangerous microbial pathogens. The present study investigates the potential of using ZIF-8 as a coating for face masks to prevent the adherence of microbial/viral entities. The results show that after 2 h of UV irradiation, a polypropylene mask coated with ZIF-8 nanostructures is capable of eliminating S. Aureus and bacteriophage MS2 with 99.99% and 95.4% efficiencies, respectively. Furthermore, low-pathogenic HCoV-OC43 coronavirus was eliminated by a ZIF-8-modified mask with 100% efficiency already after 1 h of UV irradiation. As bacteriophage MS2 and HCoV-OC43 coronavirus are commonly used surrogates of the SARS-CoV-2 virus, the revealed antiviral properties of ZIF-8 can represent an important step in designing efficient protective equipment for controlling and fighting the current COVID-19 pandemic. Publishers version

Enteroviruses are among the most common viruses causing a huge number of acute and chronic infections leading to high economic costs. Novel nontoxic antivirals that reduce the virus load in acutely infected individuals and from various surfaces are needed to efficiently combat these viruses.This review summarizes the recent findings of compounds and tools targeting the enteroviruses and host cell molecules that are crucial for virus infection. In addition, the review states the modern methods to find new targets and tools that help to understand the mechanisms of action.High-throughput molecular screens have revealed important aspects of virus life cycle in host cells and, concomitantly, some of the targets and compounds found serve as potential anti-virals combatting enterovirus infections. The risk of resistance development found for direct capsid binders lowers their usefulness, but combining them with compounds targeting evolutionarily conserved processes such as replication/translation makes them potentially a valid therapy for the future. Further automation and access to structural molecular tools such as cryo-EM and further development of, e.g. docking and simulation of large virus particles requiring heavy computation will contribute to better understanding of molecular mechanisms of action of future antivirals.

The effects of commercial enzymes (pectinases, cellulases, beta-1-3-glucanases, and pectin lyases) on the recovery of anthocyanins and polyphenols from blackcurrant press cake were studied considering two solid:solvent ratios (1:10 and 1:4 w/v). β-glucanase enabled the recovery of the highest total phenolic content – 1142 mg/100 g, and the extraction of anthocyanins was similar using all enzymes (∼400 mg/100 g). The use of cellulases and pectinases enhanced the extraction of antioxidants (DPPH − 1080 mg/100 g; CUPRAC – 3697 mg/100 g). The freeze-dried extracts presented antioxidant potential (CUPRAC, DPPH), which was associated with their biological effects in different systems: antiviral activity against both non-enveloped viruses (enterovirus coxsackievirus A-9) and enveloped coronaviruses (HCoV-OC43), and cytotoxicity towards cancer cells (A549 and HCT8). No cytotoxic effects on normal human lung fibroblast (IMR90) were observed, and no anti-inflammatory activity was detected in lipopolysaccharides-treated murine immortalised microglial cells. peerReviewed

This study aimed to characterise pressurised hot water (PHW) extracts from nonconventional sources of functional carbohydrates and phenolic compounds in terms of antioxidant capacity, antiviral activity, toxicity, and human erythrocytes’ protection antidiabetic potential. PHW extracts of Norway spruce bark (E1 + E2) and Birch sawdust (E3 + E4) contained mostly galactoglucomannan and glucuronoxylan. In contrast, samples E5 to E9 PHW extracted from Norway spruce, and Scots pine bark are rich sources of phenolic compounds. Overall, phenolic-rich extracts presented the highest inhibition of α-amylase and α-glucosidase and protection against stable non-enveloped enteroviruses. Additionally, all extracts protected human erythrocytes from hemolysis. Cell-based experiments using human cell lines (IMR90 and A549) showed extracts’ non-toxic in vitro profile. Considering the relative toxicological safety of extracts from these unconventional sources, functional carbohydrates and polyphenol-rich extracts can be obtained and further used in food models. peerReviewed

Coxsackievirus A9 (CVA9), an enterovirus, is a common cause of pediatric aseptic meningitis and neonatal sepsis. During cell entry, enterovirus capsids undergo conformational changes leading to expansion, formation of large pores, externalization of VP1 N termini, and loss of the lipid factor from VP1. Factors such as receptor binding, heat, and acidic pH can trigger capsid expansion in some enteroviruses. Here, we show that fatty acid-free bovine serum albumin or neutral endosomal ionic conditions can independently prime CVA9 for expansion and genome release. Our results showed that CVA9 treatment with albumin or endosomal ions generated a heterogeneous population of virions, which could be physically separated by asymmetric flow field flow fractionation and computationally by cryo-electron microscopy (cryo-EM) and image processing. We report cryo-EM structures of CVA9 A-particles obtained by albumin or endosomal ion treatment and a control nonexpanded virion to 3.5, 3.3, and 2.9 Å resolution, respectively. Whereas albumin promoted stable expanded virions, the endosomal ionic concentrations induced unstable CVA9 virions which easily disintegrated, losing their genome. Loss of most of the VP4 molecules and exposure of negatively charged amino acid residues in the capsid’s interior after expansion created a repulsive viral RNA-capsid interface, aiding genome release. peerReviewed

Interactions between an atomically precise gold nanocluster Au

Aqueous extracts from blackcurrant press cake (BC), Norway spruce bark (NS), Scots pine bark (SP), and sea buckthorn leaves (SB) were obtained using maceration and pressurized hot water and tested for their bioactivities. Maceration provided the extraction of higher dry matter contents, including total phenolics (TPC), anthocyanins, and condensed tannins, which also impacted higher antioxidant activity. NS and SB extracts presented the highest mean values of TPC and antioxidant activity. Individually, NS extract presented high contents of proanthocyanidins, resveratrol, and some phenolic acids. In contrast, SB contained a high concentration of ellagitannins, ellagic acid, and quercetin, explaining the antioxidant activity and antibacterial effects. SP and BC extracts had the lowest TPC and antioxidant activity. However, BC had strong antiviral efficacy, whereas SP can be considered a potential ingredient to inhibit α-amylase. Except for BC, the other extracts decreased reactive oxygen species (ROS) generation in HCT8 and A549 cells. Extracts did not inhibit the production of TNF-alpha in lipopolysaccharide-stimulated THP-1 macrophages but inhibited the ROS generation during the THP-1 cell respiratory burst. The recovery of antioxidant compounds from these by-products is incentivized for high value-added applications. peerReviewed

Viruses need to open, i.e., uncoat, in order to release their genomes for efficient replication and translation. Especially for non-enveloped viruses, such as enteroviruses, the cues leading to uncoating are less well known. The status of the virus has previously been observed mainly by transmission electron microscopy using negative staining, cryo electron microscopy, X-ray crystallography or gradient separation (reviewed in Tuthill et al., 2010, Myllynen et al., 2016, Ruokolainen et al., 2019). However, monitoring of uncoating has been limited by the lack of methods detecting dynamic changes of the virions. Here, we present a real-time fluorescence based protocol, which detects the viral genome (RNA) during various stages of uncoating in vitro, while RNA is still inside the particle that has been expanded before the actual RNA release, and when the RNA has been totally released from the viral particle. Our method allows to explore how various molecular factors may promote or inhibit virus uncoating. peerReviewed

Echovirus 30 (E30), a member of the enterovirus B species, is a major cause of viral meningitis, targeting children and adults alike. While it is a frequently isolated enterovirus and the cause of several outbreaks all over the world, surprisingly little is known regarding its entry and replication strategy within cells. In this study, we used E30 strain Bastianni (E30B) generated from an infectious cDNA clone in order to study early entry events during infection in human RD cells. E30B required the newly discovered Fc echovirus receptor (FcRn) for successful infection, but not the coxsackievirus and adenovirus receptor (CAR) or decay-accelerating factor (DAF), although an interaction with DAF was observed. Double-stranded RNA replication intermediate was generated between 2 and 3 h postinfection (p.i.), and viral capsid production was initiated between 4 and 5 h p.i. The drugs affecting Rac1 (NSC 23766) and cholesterol (filipin III) compromised infection, whereas bafilomycin A1, dyngo, U-73122, wortmannin, and nocodazole did not, suggesting the virus follows an enterovirus-triggered macropinocytic pathway rather than the clathrin pathway. Colocalization with early endosomes and increased infection due to constitutively active Rab5 expression suggests some overlap and entry to classical early endosomes. Taken together, these results suggest that E30B induces an enterovirus entry pathway, leading to uncoating in early endosomes. IMPORTANCE Echovirus 30 (E30) is a prevalent enterovirus causing regular outbreaks in both children and adults in different parts of the world. It is therefore surprising that relatively little is known of its infectious entry pathway. We set out to generate a cDNA clone and gradient purified the virus in order to study the early entry events in human cells. We have recently studied other enterovirus B group viruses, like echovirus 1 (EV1) and coxsackievirus A9 (CVA9), and found many similarities between those viruses, allowing us to define a so-called “enterovirus entry pathway.” Here, E30 is reminiscent of these viruses, for example, by not relying on acidification for infectious entry. However, despite not using the clathrin entry pathway, E30 accumulates in classical early endosomes.

Aims/hypothesis: The Coxsackie and adenovirus receptor (CAR) is a transmembrane cell-adhesion protein that serves as an entry receptor for enteroviruses and may be essential for their ability to infect cells. Since enteroviral infection of beta cells has been implicated as a factor that could contribute to the development of type 1 diabetes, it is often assumed that CAR is displayed on the surface of human beta cells. However, CAR exists as multiple isoforms and it is not known whether all isoforms subserve similar physiological functions. In the present study, we have determined the profile of CAR isoforms present in human beta cells and monitored the subcellular localisation of the principal isoform within the cells. Methods: Formalin-fixed, paraffin-embedded pancreatic sections from non-diabetic individuals and those with type 1 diabetes were studied. Immunohistochemistry, confocal immunofluorescence, electron microscopy and western blotting with isoform-specific antisera were employed to examine the expression and cellular localisation of the five known CAR isoforms. Isoform-specific qRT-PCR and RNA sequencing (RNAseq) were performed on RNA extracted from isolated human islets. Results: An isoform of CAR with a terminal SIV motif and a unique PDZ-binding domain was expressed at high levels in human beta cells at the protein level. A second isoform, CAR-TVV, was also present. Both forms were readily detected by qRT-PCR and RNAseq analysis in isolated human islets. Immunocytochemical studies indicated that CAR-SIV was the principal isoform in islets and was localised mainly within the cytoplasm of beta cells, rather than at the plasma membrane. Within the cells it displayed a punctate pattern of immunolabelling, consistent with its retention within a specific membrane-bound compartment. Co-immunofluorescence analysis revealed significant co-localisation of CAR-SIV with zinc transporter protein 8 (ZnT8), prohormone convertase 1/3 (PC1/3) and insulin, but not proinsulin. This suggests that CAR-SIV may be resident mainly in the membranes of insulin secretory granules. Immunogold labelling and electron microscopic analysis confirmed that CAR-SIV was localised to dense-core (insulin) secretory granules in human islets, whereas no immunolabelling of the protein was detected on the secretory granules of adjacent exocrine cells. Importantly, CAR-SIV was also found to co-localise with protein interacting with C-kinase 1 (PICK1), a protein recently demonstrated to play a role in insulin granule maturation and trafficking. Conclusions/interpretation: The SIV isoform of CAR is abundant in human beta cells and is localised mainly to insulin secretory granules, implying that it may be involved in granule trafficking and maturation. We propose that this subcellular localisation of CAR-SIV contributes to the unique sensitivity of human beta cells to enteroviral infection., SCOPUS: ar.j, info:eu-repo/semantics/published

According to the WHO, there is an urgent need for better control of viral diseases. Re-positioning existing safe-in-human antiviral agents from one viral disease to another could play a pivotal role in this process. Here, we reviewed all approved, investigational and experimental antiviral agents, which are safe in man, and identified 59 compounds that target at least three viral diseases. We tested 55 of these compounds against eight different RNA and DNA viruses. We found novel activities for dalbavancin against echovirus 1, ezetimibe against human immunodeficiency virus 1 and Zika virus, as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against Rift valley fever virus. Thus, the spectrum of antiviral activities of existing antiviral agents could be expanded towards other viral diseases., Highlights • 339 approved, investigational and experimental safe-in-human antivirals were identified. • 59 compounds, which target ≥3 viral diseases, were selected. • 55 of the 59 compounds were tested against 8 RNA and DNA viruses. • 7 compounds were found to possess novel antiviral activities.

A virus needs the host cell in order to replicate and produce new progeny viruses. For this, the virus takes over the host cell and modifies it to become a factory for viral proteins. Irrespective of the specific virus family, these proteins can be divided into structural and nonstructural proteins. Structural proteins are the building blocks for the new progeny virions, whereas the nonstructural proteins orchestrate the takeover of the host cell and its functions. Here, we have shown a mechanism that viruses exploit in order to regulate the host cell. We show that viral protein synthesis induces vimentin cages, which promote production of specific viral proteins that eventually control apoptosis and host cell death. This study specifies vimentin as the key regulator of these events and indicates that viral proteins have different fates in the cells depending on their association with vimentin cages., We report that several viruses from the human enterovirus group B cause massive vimentin rearrangements during lytic infection. Comprehensive studies suggested that viral protein synthesis was triggering the vimentin rearrangements. Blocking the host cell vimentin dynamics with β, β′-iminodipropionitrile (IDPN) did not significantly affect the production of progeny viruses and only moderately lowered the synthesis of structural proteins such as VP1. In contrast, the synthesis of the nonstructural proteins 2A, 3C, and 3D was drastically lowered. This led to attenuation of the cleavage of the host cell substrates PABP and G3BP1 and reduced caspase activation, leading to prolonged cell survival. Furthermore, the localization of the proteins differed in the infected cells. Capsid protein VP1 was found diffusely around the cytoplasm, whereas 2A and 3D followed vimentin distribution. Based on protein blotting, smaller amounts of nonstructural proteins did not result from proteasomal degradation but from lower synthesis without intact vimentin cage structure. In contrast, inhibition of Hsp90 chaperone activity, which regulates P1 maturation, lowered the amount of VP1 but had less effect on 2A. The results suggest that the vimentin dynamics regulate viral nonstructural protein synthesis while having less effect on structural protein synthesis or overall infection efficiency. The results presented here shed new light on differential fate of structural and nonstructural proteins of enteroviruses, having consequences on host cell survival. IMPORTANCE A virus needs the host cell in order to replicate and produce new progeny viruses. For this, the virus takes over the host cell and modifies it to become a factory for viral proteins. Irrespective of the specific virus family, these proteins can be divided into structural and nonstructural proteins. Structural proteins are the building blocks for the new progeny virions, whereas the nonstructural proteins orchestrate the takeover of the host cell and its functions. Here, we have shown a mechanism that viruses exploit in order to regulate the host cell. We show that viral protein synthesis induces vimentin cages, which promote production of specific viral proteins that eventually control apoptosis and host cell death. This study specifies vimentin as the key regulator of these events and indicates that viral proteins have different fates in the cells depending on their association with vimentin cages.

There is limited information about the molecular triggers leading to the uncoating of enteroviruses under physiological conditions. Using real-time spectroscopy and sucrose gradients with radioactively labeled virus, we show at 37°C, the formation of albumin-triggered, metastable uncoating intermediate of echovirus 1 without receptor engagement. This conversion was blocked by saturating the albumin with fatty acids. High potassium but low sodium and calcium concentrations, mimicking the endosomal environment, also induced the formation of a metastable uncoating intermediate of echovirus 1. Together, these factors boosted the formation of the uncoating intermediate, and the infectivity of this intermediate was retained, as judged by end-point titration. Cryo-electron microscopy reconstruction of the virions treated with albumin and high potassium, low sodium, and low calcium concentrations resulted in a 3.6-Å resolution model revealing a fenestrated capsid showing 4% expansion and loss of the pocket factor, similarly to altered (A) particles described for other enteroviruses. The dimer interface between VP2 molecules was opened, the VP1 N termini disordered and most likely externalized. The RNA was clearly visible, anchored to the capsid. The results presented here suggest that extracellular albumin, partially saturated with fatty acids, likely leads to the formation of the infectious uncoating intermediate prior to the engagement with the cellular receptor. In addition, changes in mono- and divalent cations, likely occurring in endosomes, promote capsid opening and genome release. IMPORTANCE There is limited information about the uncoating of enteroviruses under physiological conditions. Here, we focused on physiologically relevant factors that likely contribute to opening of echovirus 1 and other B-group enteroviruses. By combining biochemical and structural data, we show that, before entering cells, extracellular albumin is capable of priming the virus into a metastable yet infectious intermediate state. The ionic changes that are suggested to occur in endosomes can further contribute to uncoating and promote genome release, once the viral particle is endocytosed. Importantly, we provide a detailed high-resolution structure of a virion after treatment with albumin and a preset ion composition, showing pocket factor release, capsid expansion, and fenestration and the clearly visible genome still anchored to the capsid. This study provides valuable information about the physiological factors that contribute to the opening of B group enteroviruses. peerReviewed

Type B Coxsackieviruses (CVBs) are a common cause of acute and chronic myocarditis, dilated cardiomyopathy and aseptic meningitis. However, no CVB-vaccines are available for human use. We have previously produced virus-like particles (VLPs) for CVB3 with a baculovirus-insect cell production system. Here we have explored the potential of a VLP-based vaccine targeting CVB1 and describe the production of CVB1-VLPs with a scalable VLP purification method. The developed purification method consisting of tangential flow filtration and ion exchange chromatography is compatible with industrial scale production. CVB1-VLP vaccine was treated with UV-C or formalin to study whether stability and immunogenicity was affected. Untreated, UV treated and formalin treated VLPs remained morphologically intact for 12 months at 4 °C. Formalin treatment increased, whereas UV treatment decreased the thermostability of the VLP-vaccine. High neutralising and total IgG antibody levels, the latter predominantly of a Th2 type (IgG1) phenotype, were detected in female BALB/c mice immunised with non-adjuvanted, untreated CVB1-VLP vaccine. The immunogenicity of the differently treated CVB1-VLPs (non-adjuvanted) were compared in C57BL/6 J mice and animals vaccinated with formalin treated CVB1-VLPs mounted the strongest neutralising and, CVB1-specific IgG and IgG1 antibody responses. This study demonstrates that formalin treatment increases the stability and immunogenicity of CVB1-VLP vaccine and may offer a universal tool for the stabilisation of VLPs in the production of more efficient vaccines.

Echoviruses are amongst the most common causative agents of aseptic meningitis worldwide and are particularly devastating in the neonatal population, where they are associated with severe hepatitis, neurological disease, including meningitis and encephalitis, and even death. Here, we identify the neonatal Fc receptor (FcRn) as a pan-echovirus receptor. We show that loss of expression of FcRn or its binding partner beta 2 microglobulin (β2M) renders cells resistant to infection by a panel of echoviruses at the stage of virus attachment, and that a blocking antibody to β2M inhibits echovirus infection in cell lines and in primary human intestinal epithelial cells. We also show that expression of human, but not mouse, FcRn renders nonpermissive human and mouse cells sensitive to echovirus infection and that the extracellular domain of human FcRn directly binds echovirus particles and neutralizes infection. Lastly, we show that neonatal mice expressing human FcRn are more susceptible to echovirus infection by the enteral route. Our findings thus identify FcRn as a pan-echovirus receptor, which may explain the enhanced susceptibility of neonates to echovirus infections.

Type B Coxsackieviruses (CVBs) belong to the enterovirus genus, and they cause both acute and chronic diseases in humans. CVB infections usually lead to flu-like symptoms but can also result in more serious diseases such as myocarditis, aseptic meningitis and life-threatening multi-organ infections in young infants. Thus, CVBs have long been considered as important targets of future vaccines. We have previously observed CVB1 capsid disintegration and virus concentration decrease with 12-day long formalin inactivation protocol. Here a scalable ion exchange chromatography purification method was developed, and purified CVB1 was inactivated with UV-C or formalin. Virus morphology and concentration remained unchanged, when the UV (2 min) or formalin (5 days) inactivation were performed in the presence of tween80 detergent. The concentration of the native and UV inactivated CVB1 remained constant at 4 °C during a six months stability study, whereas the concentration of the formalin inactivated vaccine decreased 29% during this time. UV treatment decreased, whereas formalin treatment increased the thermal stability of the capsid. The formalin inactivated CVB1 vaccine was more immunogenic than the UV inactivated vaccine; the protective neutralizing antibody levels were higher in mice immunized with formalin inactivated vaccine. High levels of CVB1 neutralizing antibodies as well as IgG1 antibodies were detected in mice that were protected against viremia induced by experimental CVB1 infection. In conclusion, this study describes a scalable ion exchange chromatography purification method and optimized 5-day long formalin inactivation method that preserves CVB1 capsid structure and immunogenicity. Formalin treatment stabilizes the virus particle at elevated temperatures, and the formalin inactivated vaccine induces high levels of serum IgG1 antibodies (Th2 type response) and protective levels of neutralizing antibodies. Formalin inactivated CVB vaccines are promising candidates for human clinical trials.

Enteroviruses are small RNA viruses that cause diseases with various symptoms ranging from mild to severe. Enterovirus proteins are translated as a single polyprotein, which is cleaved by viral proteases to release capsid and nonstructural proteins. Here, we show that also cellular calpains have a potential role in the processing of the enteroviral polyprotein. Using purified calpains 1 and 2 in an in vitro assay, we show that addition of calpains leads to an increase in the release of VP1 and VP3 capsid proteins from P1 of enterovirus B species, detected by western blotting. This was prevented with a calpain inhibitor and was dependent on optimal calcium concentration, especially for calpain 2. In addition, calpain cleavage at the VP3-VP1 interface was supported by a competition assay using a peptide containing the VP3-VP1 cleavage site. Moreover, a mass spectrometry analysis showed that calpains can cleave this same peptide at the VP3-VP1 interface, the cutting site being two amino acids aside from 3C&rsquo, s cutting site. Furthermore, we show that calpains cannot cleave between P1 and 2A. In conclusion, we show that cellular proteases, calpains, can cleave structural proteins from enterovirus polyprotein in vitro. Whether they assist polyprotein processing in infected cells remains to be shown.

Enterovirus B species typically cause a rapid cytolytic infection leading to efficient release of progeny viruses. However, they are also capable of persistent infections in tissues, which are suggested to contribute to severe chronic states such as myocardial inflammation and type 1 diabetes. In order to understand the factors contributing to differential infection strategies, we constructed a chimera by combining the capsid proteins from fast-cytolysis-causing echovirus 1 (EV1) with nonstructural proteins from coxsackievirus B5 (CVB5), which shows persistent infection in RD cells. The results showed that the chimera behaved similarly to parental EV1, leading to efficient cytolysis in both permissive A549 and semipermissive RD cells. In contrast to EV1 and the chimera, CVB5 replicated slowly in permissive cells and showed persistent infection in semipermissive cells. However, there was no difference in the efficiency of uptake of CVB5 in A549 or RD cells in comparison to the chimera or EV1. CVB5 batches constantly contained significant amounts of empty capsids, also in comparison to CVB5's close relative CVB3. During successive passaging of batches containing only intact CVB5, increasing amounts of empty and decreasing amounts of infective capsids were produced. Our results demonstrate that the increase in the amount of empty particles and the lowering of the amount of infective particles are dictated by the CVB5 structural proteins, leading to slowing down of the infection between passages. Furthermore, the key factor for persistent infection is the small amount of infective particles produced, not the high number of empty particles that accumulate. IMPORTANCE Enteroviruses cause several severe diseases, with lytic infections that lead to rapid cell death but also persistent infections that are more silent and lead to chronic states of infection. Our study compared a cytolytic echovirus 1 infection to persistent coxsackievirus B5 infection by making a chimera with the structural proteins of echovirus 1 and the nonstructural proteins of coxsackievirus B5. Coxsackievirus B5 infection was found to lead to the production of a high number of empty viruses (empty capsids) that do not contain genetic material and are unable to continue the infection. Coinciding with the high number of empty capsids, the amount of infective virions decreased. This characteristic property was not observed in the constructed chimera virus, suggesting that structural proteins are in charge of these phenomena. These results shed light on the mechanisms that may cause persistent infections. Understanding events leading to efficient or inefficient infections is essential in understanding virus-caused pathologies.

The current methods to study the distribution and dynamics of viral RNA molecules inside infected cells are not ideal, as electron microscopy and immunohistochemistry can only detect mature virions, and quantitative real-time PCR does not reveal localized distribution of RNAs. We demonstrated here the branched DNA in situ hybridization (bDNA ISH) technology to study both the amount and location of the emerging &minus, RNA and +RNA during acute and persistent enterovirus infections. According to our results, the replication of the viral RNA started 2&ndash, 3 h after infection and the translation shortly after at 3&ndash, 4 h post-infection. The replication hotspots with newly emerging &minus, RNA were located quite centrally in the cell, while the +RNA production and most likely virion assembly took place in the periphery of the cell. We also discovered that the pace of replication of &minus, RNA and +RNA strands was almost identical, and &minus, RNA was absent during antiviral treatments. ViewRNA ISH with our custom probes also showed a good signal during acute and persistent enterovirus infections in cell and mouse models. Considering these results, along with the established bDNA FISH protocol modified by us, the effects of antiviral drugs and the emergence of enterovirus RNAs in general can be studied more effectively.

Fluorescent probes for sensing nucleic acids have found widespread use in the field of cell and molecular biology. However, probes combined with potential for post-synthetic conjugation, e.g. for intra-endosomal measurements of RNA, are unavailable. Herein we developed cyanine dyes that can be conjugated to viral capsid or other targets. First, we solved the crystal structure of SYBR Green II. The structural elucidation of this commonly used RNA probe provided the basis for synthesizing similar molecules with much desired function for post-synthetic conjugation. To address this need, cyanine dyes were prepared using an alternative synthesis protocol. All studied compounds showed considerable brightness upon binding to nucleic acids. However, regardless of the common chromophore on the dyes, the observed fluorescence emission intensities varied significantly, where methyl-substituted dye 1 gave values higher than SYBR Green II, whereas compounds 2–5 containing undecyl spacers had lower values. Studying the structure-activity relationship revealed the longer alkyl chains to induce slight perturbation in dye intercalation, as well as demand larger binding area on the nucleic acid lattice, explaining these differences. To study the potential biological use of the dyes, the RNA genome of enterovirus echovirus 1 was studied in vitro with the probes. A novel method employing the low binding space requirement of 1 was developed to determine the single-to-double-stranded RNA ratio of a sample, whereas compound 4 was covalently bound to the viral capsid and used successfully to monitor the viral RNA release from within the capsid. The presented results open new possibilities for preparation and use of SYBR Green-based nucleic acid probes to further apply these compounds for increasingly demanding targeting in biological contexts.

Enteroviruses (EVs) are common RNA viruses that cause diseases ranging from rash to paralytic poliomyelitis. For example, EV-A and EV-C viruses cause hand-foot and mouth disease and EV-B viruses cause encephalitis and myocarditis, which can result in severe morbidity and mortality. While new vaccines and treatments for EVs are under development, methods for studying and diagnosing EV infections are still limited and therefore new diagnostic tools are required. Our aim was to produce and characterize new antibodies that work in multiple applications and detect EVs in tissues and in vitro. Rats were immunized with Coxsackievirus B1 capsid protein VP1 and hybridomas were produced. Hybridoma clones were selected based on their reactivity in different immunoassays. The most promising clone, 3A6, was characterized and it performed well in multiple techniques including ELISA, immunoelectron microscopy, immunocyto- and histochemistry and in Western blotting, detecting EVs in infected cells and tissues. It recognized several EV-Bs and also the EV-C representative Poliovirus 3, making it a broad-spectrum EV specific antibody. The 3A6 rat monoclonal antibody can help to overcome some of the challenges faced with commonly used EV antibodies: it enables simultaneous use of mouse-derived antibodies in double staining and it is useful in murine models.

Viral infections are amongst the most common diseases affecting people worldwide. New viruses emerge all the time and presently we have limited number of vaccines and only few antivirals to combat viral diseases. Fungi represent a vast source of bioactive molecules, which could potentially be used as antivirals in the future. Here, we have summarized the current knowledge of fungi as producers of antiviral compounds and discuss their potential applications. In particular, we have investigated how the antiviral action has been assessed and what is known about the molecular mechanisms and actual targets. Furthermore, we highlight the importance of accurate fungal species identification on antiviral and other natural products studies. peerReviewed

Visualization and tracking of viruses without compromising their functionality is crucial in order to understand virus targeting to cells and tissues, and to understand the subsequent subcellular steps leading to virus uncoating and replication. Enteroviruses are important human pathogens causing a vast number of acute infections, and are also suggested to contribute to the development of chronic diseases like type I diabetes. Here, we demonstrate a novel method to target site-specifically the hydrophobic pocket of enteroviruses. A probe, a derivative of Pleconaril, was developed and conjugated to various labels that enabled the visualization of enteroviruses under light and electron microscopes. The probe mildly stabilized the virus particle by increasing the melting temperature by 1-3 degrees, and caused a delay in the uncoating of the virus in the cellular endosomes, but could not however inhibit the receptor binding, cellular entry or infectivity of the virus. The hydrophobic pocket binding moiety of the probe was shown to bind to echovirus 1 particle by STD and tr-NOESY NMR methods. Furthermore, binding to echovirus 1 and Coxsackievirus A9, and to a lesser extent to Coxsackie virus B3 was verified by using a gold nanocluster labeled probe by TEM analysis. Molecular modelling suggested that the probe fits the hydrophobic pockets of EV1 and CVA9, but not of CVB3 as expected, correlating well with the variations in the infectivity and stability of the virus particles. EV1 conjugated to the fluorescent dye labeled probe was efficiently internalized into the cells. The virus-fluorescent probe conjugate accumulated in the cytoplasmic endosomes and caused infection starting from 6 hours onwards. Remarkably, before and during the time of replication, the fluorescent probe was seen to leak from the virus-positive endosomes and thus separate from the capsid proteins that were left in the endosomes. These results suggest that, like the physiological hydrophobic content, the probe may be released upon virus uncoating. Our results collectively thus show that the gold and fluorescently labeled probes may be used to track and visualize the studied enteroviruses during the early phases of infection opening new avenues to follow virus uncoating in cells.

In addition to vaccines, noninfectious virus-like particles (VLPs) that mimic the viral capsid show an attractive possibility of presenting immunogenic epitopes or targeting molecules on their surface. Here, functionalization of norovirus-derived VLPs by simple non-covalent conjugation of various molecules is shown. By using the affinity between a surface-exposed polyhistidine-tag and multivalent tris-nitrilotriacetic acid (trisNTA), fluorescent dye molecules and streptavidin–biotin conjugated to trisNTA are displayed on the VLPs to demonstrate the use of these VLPs as easily modifiable nanocarriers as well as a versatile vaccine platform. The VLPs are able to enter and deliver surface-displayed fluorescent dye into HEK293T cells via a surface-attached cell internalization peptide (VSV-G). The ease of manufacturing, the robust structure of these VLPs, and the straightforward conjugation provide a technology, which can be adapted to various applications in biomedicine.