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SARS-CoV-2 and HCoV-OC43 belong to the same β genus of the Coronaviridae family. SARS-CoV-2 was responsible for the recent COVID-19 pandemic, and HCoV-OC43 is the etiological agent of mild upper respiratory tract infections. SARS-COV-2 and HCoV-OC43 co-infections were found in children with respiratory symptoms during the COVID-19 pandemic. The two β-coronaviruses share a high degree of homology between the 3CLpro active sites, so much so that the safer HCoV-OC43 has been suggested as a tool for the identification of new anti-SARS-COV-2 agents. Compounds 5 and 24 inhibited effectively both Wuhan and British SARS-CoV-2 patient isolates in Vero E6 cells and the HCoV-OC43 in MRC-5 cells at low micromolar concentrations. The inhibition was apparently exerted via targeting the 3CLpro active sites of both viruses. Compounds 5 and 24 at 100 μM inhibited the SARS-CoV-2 3CLpro activity of 61.78 and 67.30%, respectively. These findings highlight 5 and 24 as lead compounds of a novel class of antiviral agents with the potential to treat SARS-COV-2 and HCoV-OC43 infections.

Infectious and toxicological risks are the main potential hazards that operators of Human Milk Banks (HMBs) encounter and must eliminate. HMBs are trying to implement procedures that allow to manage and sanitize human milk without altering significantly its nutritional and biologically protective components, obtaining a product characterized by a valid balance between safety and biological quality. The history of human milk processing is linked to the origins of HMBs themselves. And although other forms of sterilization were used originally, pasteurization soon became the recognized most effective means for sanitizing milk: all the milk that arrives at the HMB must be pasteurized. Holder pasteurization (HoP) is the most used methodology, and it is performed using low temperature and long time (+62.5°C for 30 min). With HoP some bioactive milk components are lost to varying degrees, but many other precious bioactive compounds are completely or partially preserved. To improve the quality of human milk processed by HMBs, maintaining in the meantime the same microbiological safety offered by HoP, new technologies are under evaluation. At present, High-Temperature Short-Time pasteurization (HTST) and High-Pressure Processing are the most studied methodologies. HTST is already utilized in some HMBs for daily practical activity and for research purposes. They seem to be superior to HoP for a better preservation of some nutritional and biologically protective components. Freeze-drying or lyophilization may have advantages for room temperature storage and transportation. The aim of this study is to evaluate the advancement regarding the processing of DHM with a literature search from 2019 to 2022. The effects of the new technologies on safety and quality of human milk are presented and discussed. The new technologies should assure microbiological safety of the final product at least at the same level as optimized HoP, with an improved preservation of the nutritional and bioactive components of raw human milk., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Moro, Girard, Peila, Garcia, Escuder-Vieco, Keller, Cassidy, Bertino, Boquien, Buffin, Calvo, Gaya, Gebauer, Lamireau, Lembo, Picaud, Wesolowska, Arslanoglu, Cavallarin and Giribaldi.)

Human respiratory viruses have an enormous impact on national health systems, societies, and economy due to the rapid airborne transmission and epidemic spread of such pathogens, while effective specific antiviral drugs to counteract infections are still lacking. Here, we identified two Keggin-type polyoxometalates (POMs), [TiW 11 CoO 40 ] 8- (TiW 11 Co) and [Ti 2 PW 10 O 40 ] 7- (Ti 2 PW 10 ), endowed with broad-spectrum activity against enveloped and non-enveloped human respiratory viruses, i.e., coronavirus (HCoV-OC43), rhinovirus (HRV-A1), respiratory syncytial virus (RSV-A2), and adenovirus (AdV-5). Ti 2 PW 10 showed highly favorable selectivity indexes against all tested viruses (SIs >700), and its antiviral potential was further investigated against human coronaviruses and rhinoviruses. This POM was found to inhibit replication of multiple HCoV and HRV strains, in different cell systems. Ti 2 PW 10 did not affect virus binding or intracellular viral replication, but selectively inhibited the viral entry. Serial passaging of virus in presence of the POM revealed a high barrier to development of Ti 2 PW 10 -resistant variants of HRV-A1 or HCoV-OC43. Moreover, Ti 2 PW 10 was able to inhibit HRV-A1 production in a 3D model of the human nasal epithelium and, importantly, the antiviral treatment did not determine cytotoxicity or tissue damage. A mucoadhesive thermosensitive in situ hydrogel formulation for nasal delivery was also developed for Ti 2 PW 10 . Overall, good biocompatibility on cell lines and human nasal epithelia, broad-spectrum activity, and absence of antiviral resistance development reveal the potential of Ti 2 PW 10 as an antiviral candidate for the development of a treatment of acute respiratory viral diseases, warranting further studies to identify the specific target/s of the polyanion and assess its clinical potential., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

During the SARS-CoV-2 pandemic, many countries established wastewater (WW) surveillance to objectively monitor the level of infection within the population. As new variants continue to emerge, it has become clear that WW surveillance is an essential tool for the early detection of variants. The EU Commission published a recommendation suggesting an approach to establish surveillance of SARS-CoV-2 and its variants in WW, besides specifying the methodology for WW concentration and RNA extraction. Therefore, different groups have approached the issue with different strategies, mainly focusing on WW concentration methods, but only a few groups highlighted the importance of prefiltering WW samples and/or purification of RNA samples. Aiming to obtain high-quality sequencing data allowing variants detection, we compared four experimental conditions generated from the treatment of: i) WW samples by WW filtration and ii) the extracted RNA by DNase treatment, purification and concentration of the extracted RNA. To evaluate the best condition, the results were assessed by focusing on several sequencing parameters, as the outcome of SARS-CoV-2 sequencing from WW is crucial for variant detection. Overall, the best sequencing result was obtained by filtering the WW sample. Moreover, the present study provides an overview of some sequencing parameters to consider when optimizing a method for monitoring SARS-CoV-2 variants from WW samples, which can also be applied to any sample preparation methodology., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Robotto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The bacterial genotoxin colibactin promotes colorectal cancer (CRC) tumorigenesis, but systematic assessment of its impact on DNA repair is lacking, and its effect on response to DNA-damaging chemotherapeutics is unknown. We find that CRC cell lines display differential response to colibactin on the basis of homologous recombination (HR) proficiency. Sensitivity to colibactin is induced by inhibition of ATM, which regulates DNA double-strand break repair, and blunted by HR reconstitution. Conversely, CRC cells chronically infected with colibactin develop a tolerant phenotype characterized by restored HR activity. Notably, sensitivity to colibactin correlates with response to irinotecan active metabolite SN38, in both cell lines and patient-derived organoids. Moreover, CRC cells that acquire colibactin tolerance develop cross-resistance to SN38, and a trend toward poorer response to irinotecan is observed in a retrospective cohort of CRCs harboring colibactin genomic island. Our results shed insight into colibactin activity and provide translational evidence on its chemoresistance-promoting role in CRC., Competing Interests: Declaration of interests The authors declare the following competing interests, which are unrelated to the results of the study. A.B. reports receiving commercial research grants from Neophore, AstraZeneca, and Boehringer; he is an advisory board member/unpaid consultant for Inivata and Neophore, holds ownership interest in Neophore, and is an advisory board member/consultant for Illumina, Guardant Health, Inivata, and Roche/Genentech Global CRC. F.D.N. received honoraria from Pierre Fabre. L.T. has received research grants from Menarini, Merck KGaA, Merus, Pfizer, Servier, and Symphogen. S.A. acted as consultant for MSD Italia outside the submitted work and has a patent (102022000007535) pending. A.S.-B. is an advisory board member for Amgen, Bayer, Novartis, Pierre Fabre, and Servier. S.S. is an advisory board member for Agenus, AstraZeneca, Bayer, Bristol Myers Squibb, CheckmAb, Daiichi Sankyo, Guardant Health, Menarini, Merck, Novartis, Roche-Genentech, and Seagen., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The indoor air quality should be better controlled and improved to avoid numerous health issues. Even if different devices are developed for air filtration, the proliferation of microorganisms under certain conditions must be controlled. For this purpose, a silver nanocluster/silica composite coating was deposited via a cosputtering technique onto fiber glass and polymeric based substrates. The aim of this work is focused on the evaluation of the antibacterial and antiviral effects of the developed coating. The preliminary results of the compositional and morphological tests showed an evenly distributed coating on filters surfaces. Several antibacterial tests were performed, confirming strong effect both in qualitative and quantitative methods, against S. epidermidis and E. coli . To understand if the coating can stop the proliferation of bacteria colonies spread on it, simulation of everyday usage of filters was performed, nebulizing bacteria solution with high colonies concentration and evaluating the inhibition of bacteria growth. Additionally, a deep understanding of the virucidal action and mechanism of Ag nanoclusters of the coating was performed. The effect of the coating both in aqueous medium and in dry methods was evaluated, in comparison with analysis on ions release. The virucidal performances are assessed against the human coronavirus OC43 strain (HCoV-OC43).

Despite the increasing body of evidence supporting the use of simulation in medicine, a question remains: when should we introduce it into the medical school's curriculum? We present the experience and future perspectives of the MD program in Medicine and Surgery of University of Turin-MedInTo. Since its launch, MedInTo has been dedicated to integrating innovative teaching approaches at the early stages into the medical curriculum. Herewith, we describe a case-based approach for our activities, which includes the utilization of simulation for emergency medical care training for students and the integration of virtual and augmented reality technology. Dedicated surgical training activities using virtual-augmented reality and life-like simulator for students are also described., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Lembo, Abate Daga, Calì, Garbossa, Manfredi, Odetto, Ostacoli, Paccotti, Raimondo, Reimondo and Sciascia.)

Background: While the prevalence of antiphospholipid antibodies (aPL) in venous and arterial thrombotic events had already been estimated by previous studies, the prevalence of aPL in subjects with Thrombotic Microangiopathy (TMA) is still not fully elucidated. Thus, we conducted a systematic review to estimate the frequency of aPL in subjects with biopsy-proven renal TMA., Methods: We conducted in the PubMed database a search for English-language studies investigating the presence of aPL in subjects with biopsy-proven renal TMA from January 1985 to December 2022. Keywords used in the search included: 'antiphospholipid syndrome', 'antiphospholipid antibodies' and 'thrombotic microangiopathy'. Cohorts of HUS patients were excluded due to the risk of over-estimating the prevalence of aPL in these populations. The median frequency for positive aPL including anticardiolipin antibodies (aCL), antibodies against β2-glycoprotein-I (anti-β2GPI) and lupus anticoagulant (LA) was then calculated., Results: 522 articles were identified through the literature search. Six studies, assessing the prevalence of aPL in 211 subjects with renal TMA, were retrieved. The overall aPL prevalence was estimated as 24.4% (range 22-56). The estimated prevalence of aCL (IgG/IgM), anti-β2GPI, (IgG/IgM) and LA was 4.0% (range 3-27), 4.0% (range 3-16) and 18.9% (range 13-25), respectively. APS was diagnosed in 16.3% (range 11-29) of the patients. Of note, a high level of heterogeneity was observed when comparing the reported aPL profiles for each study., Conclusions: This comprehensive systematic analysis of studies investigating the prevalence of aPL in renal TMA showed that, despite the high heterogeneity of the included studies, aPL are present in about one case out of four renal-TMA cases., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Human milk (HM) is considered the best source of nutrition for infant growth and health. This nourishment is unique and changes constantly during lactation to adapt to the physiological needs of the developing infant. It is also recognized as a potential route of transmission of some viral pathogens although the presence of a virus in HM rarely leads to a disease in an infant. This intriguing paradox can be explained by considering the intrinsic antiviral properties of HM. In this comprehensive and schematically presented review, we have described what viruses have been detected in HM so far and what their potential transmission risk through breastfeeding is. We have provided a description of all the antiviral compounds of HM, along with an analysis of their demonstrated and hypothesized mechanisms of action. Finally, we have also analyzed the impact of HM pasteurization and storage methods on the detection and transmission of viruses, and on the antiviral compounds of HM. We have highlighted that there is currently a deep knowledge on the potential transmission of viral pathogens through breastfeeding and on the antiviral properties of HM. The current evidence suggests that, in most cases, it is unnecessarily to deprive an infant of this high-quality nourishment and that the continuation of breastfeeding is in the best interest of the infant and the mother., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Valacyclovir (VACV) was developed as a prodrug of the most common anti-herpetic drug Acyclovir (ACV), aiming to enhance its bioavailability. Nevertheless, prolonged VACV oral treatment may lead to the development of important side effects. Nanotechnology-based formulations for vaginal administration represent a promising approach to increase the concentration of the drug at the site of infection, limiting systemic drug exposure and reducing systemic toxicity. In this study, VACV-loaded nanodroplet (ND) formulations, optimized for vaginal delivery, were designed. Cell-based assays were then carried out to evaluate the antiviral activity of VACV loaded in the ND system. The chitosan-shelled ND exhibited an average diameter of about 400 nm and a VACV encapsulation efficiency of approximately 91% and was characterized by a prolonged and sustained release of VACV. Moreover, a modification of chitosan shell with an anionic cyclodextrin, sulfobutyl ether β-cyclodextrin (SBEβCD), as a physical cross-linker, increased the stability and mucoadhesion capability of the nanosystem. Biological experiments showed that SBEβCD-chitosan NDs enhanced VACV antiviral activity against the herpes simplex viruses type 1 and 2, most likely due to the long-term controlled release of VACV loaded in the ND and an improved delivery of the drug in sub-cellular compartments.

Background: There is extensive evidence that Holder pasteurization (HoP) (30 min at 62.5 °C) has harmful effects on the bioactivities of human milk (HM). We previously demonstrated that lowering HoP temperature is sufficient to inactivate Cytomegalovirus (HCMV). Here, we analyzed the effect of lowering time/temperature on the antiviral activity against HCMV and IgA levels of HM., Methods: Eighty HM samples from five mothers were pasteurized in a range of temperature (62.5-56 °C) and time (40-10 min) in a conventional setting of Human Milk Bank. Unpasteurized HM from each mother was used as control. The samples were assayed against HCMV-AD169 strain in cell cultures and IgA levels were determined by ELISA., Results: All HM samples exhibited anti-HCMV activity, to a different extent. An improvement of antiviral activity was observed in samples treated at 60, 58 and 56 °C compared to those at 62.5 °C, with ID 50 values near those of unpasteurized milk. Similarly, better retention in IgA levels was observed by reducing the temperature of treatment., Conclusions: We demonstrated that a 2.5 °C reduction of heat treatment significantly preserved the IgA content and fully restored the anti-HCMV activity of HM, supporting this variant of HoP as a valid alternative to preserve HM bioactivities., Impact: This work questions the standard HoP and opens the debate on whether the pasteurization temperature commonly used in Human Milk Banks should be lowered to better preserve the biological components of the milk. A reduction of HoP temperature at 60 °C determined a significant preservation of anti-HCMV activity and IgA content of donor HM, compared to standard HoP. This alternative HoP is highly feasible compared to other substitute pasteurization techniques, since it would employ the same pasteurizer equipment found in most Human Milk Banks., (© 2023. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Three gallium(III)- and thallium(III)-containing polyoxopalladates (POPs) have been synthesized and structurally characterized in the solid state and in solution, namely, the phosphate-capped 12-palladate nanocubes [XPd 12 O 8 (PO 4 ) 8 ] 13- (X = Ga III , GaPd 12 P 8 ; X = Tl III , TlPd 12 P 8 ) and the 23-palladate double-cube [Tl 2 III Pd 23 P 14 O 70 (OH) 2 ] 20- ( Tl 2 Pd 23 P 14 ). The cuboid POPs, GaPd 12 P 8 and TlPd 12 P 8 , are solution stable as verified by the respective 31 P, 71 Ga, and 205 Tl nuclear magnetic resonance (NMR) spectra. Of prime interest, the spin-spin coupling schemes allowed for an intimate study of the solution behavior of the Tl III -containing POPs via a combination of 31 P and 205 Tl NMR, including the stoichiometry of the major fragments of Tl 2 Pd 23 P 14 . Moreover, biological studies demonstrated the antitumor and antiviral activity of GaPd 12 P 8 and TlPd 12 P 8 , which were validated to be as efficient as cis -platinum against human melanoma and acute promyelocytic leukemia cells. Furthermore, GaPd 12 P 8 and TlPd 12 P 8 exerted inhibitory activity against two herpetic viruses, HSV-2 and HCMV, in a dose-response manner.

This paper describes a novel nanoformulation for dual MRI/US in vivo monitoring of drug delivery/release. The nanosystem was made of a perfluoropentane core coated with phospholipids stabilized by glycol chitosan crosslinked with triphosphate ions, and it was co-loaded with the prodrug prednisolone phosphate (PLP) and the structurally similar MRI agent Gd-DTPAMA-CHOL. Importantly, the in vitro release of PLP and Gd-DTPAMA-CHOL from the nanocarrier showed similar profiles, validating the potential impact of the MRI agent as an imaging reporter for the drug release. On the other hand, the nanobubbles were also detectable by US imaging both in vitro and in vivo. Therefore, the temporal evolution of both MRI and US contrast after the administration of the proposed nanosystem could report on the delivery and the release kinetics of the transported drug in a given lesion.

Genital herpes, most frequently caused by herpes simplex virus 2 (HSV-2) infection, is one of the most prevalent sexually transmitted infections. The current rationale for the treatment of HSV-2 infection involves nucleoside analogs (e.g. acyclovir) to suppress reactivation. Enzymatic oxysterols are endogenous 27-carbon atoms molecules produced by enzymatic cholesterol oxidation, and recently emerged as a broad-spectrum host targeting antivirals. In this study, we screened selected members of an in-house synthesized library of oxysterol analogs for their activity against HSV-2, identifying three compounds, named PFM064, PFM067, and PFM069, endowed with 50% effective concentrations (EC 50 ) in the micromolar range, without exerting any apparent cytotoxicity. Moreover, the results obtained showed the ability of the novel derivatives to inhibit both cell-to-cell fusion induced by HSV-2, and the production of an intracellular viral progeny. Further experiments performed with PFM067 (which was selected for more-in-depth studies as the most effective synthetic analog) showed that these molecules act in a late stage of HSV-2 replicative cycle, by sequestering viral glycoproteins in the Golgi compartment, and likely inhibiting the nuclear egress of neo-synthetized viral capsids. Taken together, these results point to PFM067 as a promising chemical scaffold for the development of novel herpetic antivirals., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Andrea Civra, David Lembo, Giuseppe Poli are the founders of Panoxyvir Ltd., a start-up developing the use of specific oxysterols as broad-spectrum antiviral agents. All other authors: none to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background and Objectives: Long lasting immune response to anti-SARS-CoV-2 vaccination in people with Multiple Sclerosis (pwMS) is still largely unexplored. Our study aimed at evaluating the persistence of the elicited amount of neutralizing antibodies (Ab), their activity and T cell response after three doses of anti-SARS-CoV-2 vaccine in pwMS., Methods: We performed a prospective observational study in pwMS undergoing SARS-CoV-2 mRNA vaccinations. Anti-Region Binding Domain (anti-RBD) of the spike (S) protein immunoglobulin G (IgG) titers were measured by ELISA. The neutralization efficacy of collected sera was measured by SARS-CoV-2 pseudovirion-based neutralization assay. The frequency of Spike-specific IFNγ-producing CD4+ and CD8+ T cells was measured by stimulating Peripheral Blood Mononuclear Cells (PBMCs) with a pool of peptides covering the complete protein coding sequence of the SARS-CoV-2 S., Results: Blood samples from 70 pwMS (11 untreated pwMS, 11 under dimethyl fumarate, 9 under interferon-γ, 6 under alemtuzumab, 8 under cladribine, 12 under fingolimod and 13 under ocrelizumab) and 24 healthy donors were collected before and up to six months after three vaccine doses. Overall, anti-SARS-CoV-2 mRNA vaccine elicited comparable levels of anti-RBD IgGs, neutralizing activity and anti-S T cell response both in untreated, treated pwMS and HD that last six months after vaccination. An exception was represented by ocrelizumab-treated pwMS that showed reduced levels of IgGs (p<0.0001) and a neutralizing activity under the limit of detection (p<0.001) compared to untreated pwMS. Considering the occurrence of a SARS-CoV-2 infection after vaccination, the Ab neutralizing efficacy (p=0.04), as well as CD4+ (p=0.016) and CD8+ (p=0.04) S-specific T cells, increased in treated COVID+ pwMS compared to uninfected treated pwMS at 6 months after vaccination., Discussion: Our follow-up provides a detailed evaluation of Ab, especially in terms of neutralizing activity, and T cell responses after anti-SARS-CoV-2 vaccination in MS context, over time, considering a wide number of therapies, and eventually breakthrough infection. Altogether, our observations highlight the vaccine response data to current protocols in pwMS and underline the necessity to carefully follow-up anti-CD20- treated patients for higher risk of breakthrough infections. Our study may provide useful information to refine future vaccination strategies in pwMS., Competing Interests: MM received personal compensations for advisory boards and travel grants from Novartis, Sanofi-Genzyme; SR received personal compensations for public speaking and travel grants from Sanofi-Genzyme and Merck Serono; MC received personal compensations for advisory boards, public speaking, editorial commitments or travel grants from Biogen Idec, Merck Serono, Fondazione Serono, Novartis, Pomona, Sanofi-Genzyme and Teva. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Maglione, Francese, Arduino, Rosso, Matta, Rolla, Lembo and Clerico.)

New antiviral drugs and new preventive antiviral strategies are a target of intense scientific interest. Thanks to their peculiar properties, nanomaterials play an important role in this field, and, in particular, among metallic materials, silver nanoparticles were demonstrated to be effective against a wide range of viruses, in addition to having a strong antibacterial effect. Although the mechanism of antiviral action is not completely clarified, silver nanoparticles can directly act on viruses, and on their first steps of interaction with the host cell, depending on several factors, such as size, shape, functionalization and concentration. This review provides an overview of the antiviral properties of silver nanoparticles, along with their demonstrated mechanisms of action and factors mainly influencing their properties. In addition, the fields of potential application are analyzed, demonstrating the versatility of silver nanoparticles, which can be involved in several devices and applications, including biomedical applications, considering both human and animal health, environmental applications, such as air filtration and water treatment, and for food and textile industry purposes. For each application, the study level of the device is indicated, if it is either a laboratory study or a commercial product., Competing Interests: The authors declare no conflicts of interest.

Zika virus (ZIKV) and Usutu virus (USUV) are two emerging flaviviruses mostly transmitted by mosquitos. ZIKV is associated with microcephaly in newborns and the less-known USUV, with its reported neurotropism and its extensive spread in Europe, represents a growing concern for human health. There is still no approved vaccine or specific antiviral against ZIKV and USUV infections. The main goal of this study is to investigate the anti-ZIKV and anti-USUV activity of a new library of compounds and to preliminarily investigate the mechanism of action of the selected hit compounds in vitro. Two potent anti-ZIKV and anti-USUV agents, namely ZDL-115 and ZDL-116, were discovered, both presenting low cytotoxicity, cell-line independent antiviral activity in the low micromolar range and ability of reducing viral progeny production. The analysis of the structure-activity relationship (SAR) revealed that introduction of 2-deoxyribose to 3-arene was fundamental to enhance the solubility and improve the antiviral action. Additionally, we demonstrated that ZDL-115 and ZDL-116 are significantly active against both viruses when added on cells for at least 24 h prior to viral inoculation or immediately post-infection. The docking analysis showed that ZDL-116 could target the host vitamin D receptor (VDR) and viral proteins. Future experiments will be focused on compound modification to discover analogues that are more potent and on the clarification of the mechanism of action and the specific drug target. The discovery and the development of a novel anti-flavivirus drug will have a significant impact in a context where there are no fully effective antiviral drugs or vaccines for most flaviviruses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

The etiological complexity of Behçet syndrome (BS), an immune-mediated rare form of vasculitis characterized by multi-organ involvement, is still elusive due to an incomplete understanding of the synergy between genetic susceptibility, environmental triggers, and an abnormal immune response. Long-standing theories regarding the origins of BS include the involvement of infectious organisms supporting an aberrant immunological response through different mechanisms, including molecular mimicry. Additionally, it has been demonstrated that the BS phenotypes are linked to oral and gut microbiome dysbiosis, which is a dynamic reservoir of millions of microbes containing proteins and metabolites that can mimic the autoantigens. Infections, including viral pathogens, could potentially trigger the inflammation and symptoms of BS. In this review, we aim to describe the available evidence on the cross-talk between BS and infections in order to discuss potential clinical implications and possible therapeutic targets., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Cyclodextrins and cyclodextrin derivatives were demonstrated to improve the antiviral potency of numerous drugs, but also to be endowed with intrinsic antiviral action. They are suitable building blocks for the synthesis of functionalized polymer structures with potential antiviral activity. Accordingly, four water-soluble hyper-branched beta cyclodextrin (βCD)-based anionic polymers were screened against herpes simplex virus (HSV-2), respiratory syncytial virus (RSV), rotavirus (HRoV), and influenza virus (FluVA). They were characterized by FTIR-ATR, TGA, elemental analyses, zeta-potential measurements, and potentiometric titrations, while the antiviral activity was investigated with specific in vitro assays. The polymer with the highest negative charge, pyromellitic dianhydride-linked polymer (P&#95;PMDA), showed significant antiviral action against RSV and HSV-2, by inactivating RSV free particles and by altering HSV-2 binding to the cell. The polymer fraction with the highest molecular weight showed the strongest antiviral activity and both P&#95;PMDA and its active fractions were not toxic for cells. Our results suggest that the polymer virucidal activity against RSV can be exploited to produce new antiviral materials to counteract the virus dissemination through the air or direct contact. Additionally, the strong HSV-2 binding inhibition along with the water solubility of P&#95;PMDA and the acyclovir complexation potential of βCD are attractive features for developing new therapeutic topical options against genital HSV-2 infection.

The genetic plasiticity of viruses is one of the main obstacles to the development of antivirals. The aim of this study has been to assess the ability of two physiologic oxysterols and host-targeting antivirals - namely 25- and 27-hydroxycholesterol (25OHC and 27OHC) - to select resistant strains, using human rhinovirus (HRV) as a challenging model of a viral quasispecies. Moreover, we selected 27OHC for further studies aimed at exploring its potential for the development of antiviral drugs. The results obtained with clonal or serial passage approaches show that 25OHC and 27OHC do not select HRV oxysterol-resistant variants. Moreover, we demonstrate the ability of 27OHC to inhibit the yield of HRV in 3D in vitro fully reconstituted human nasal and bronchial epithelia from cystic fibrosis patients and prevent virus-induced cilia damage. The promising antiviral activity of 27OHC and its competitive advantages over direct-acting antivirals, make this molecule a suitable candidate for further studies to explore its clinical potential., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: The antiviral role of glycosaminoglycans in human milk (HM-GAGs) has been poorly investigated. They are highly sulfated polysaccharides, which were proposed to act as decoy receptors according to their structure. The aim of this study is to evaluate the antiviral potential and the mechanism of action of total and individual HM-GAGs against three pediatric clinically relevant viruses: respiratory syncytial virus (RSV), cytomegalovirus (HCMV), and rotavirus., Methods: HM-GAGs were isolated from HM and a library of individual GAGs, structurally related to HM-GAGs, was prepared. The antiviral activity of HM-GAGs and the impact of thermal treatment were investigated in vitro by specific antiviral assays., Results: We demonstrated that HM-GAGs are endowed with anti-HCMV and anti-RSV activity and that they act by altering virus attachment to cell. We clarified the contribution of individual HM-GAGs, showing a specific structure-related activity. We did not observe any alteration of HM-GAG antiviral activity after thermal treatment., Conclusions: We showed that HM-GAGs contribute to the overall antiviral activity of HM, likely exerting a synergic action with other HM antiviral agents. HM-GAGs can now be added to the list of endogenous factors that may reduce breast-milk-acquired HCMV symptomatic infections and protecting infants from respiratory tract infections by RSV., Impact: HM-GAGs have been poorly investigated for their antiviral action so far. We demonstrated that HM-GAGs are endowed with significant anti-HCMV and anti-RSV activity and that they are able to alter virus binding to the cell. The contribution of individual HM-GAGs is mainly exerted by the FMHep and is not based on a simple charge interaction between the virus and sulfate groups but involves a specific GAG structural configuration. Our results contribute to identifying the multiple factors synergically acting in mediating HM antiviral properties and to clarifying their specific mechanism of action., (© 2022. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

The experience gained over the last hundred years clearly indicates that two groups of viruses represent the main risk for the development of highly transmissible epidemics and pandemics in the human species: influenza viruses and coronaviruses (CoV). Although the search for viruses with pandemic potential in the environment may have an important predictive and monitoring role, it is still based on empirical methodologies, mostly resulting from the clinic and not fully validated for environmental matrices. As far as the SARS-CoV-2 pandemic, currently underway, is concerned, environmental monitoring activities aiming at checking the presence of SARS-CoV-2 in wastewater can be extremely useful to predict and check the diffusion of the disease. For this reason, the present study aims at evaluating the SARS-CoV-2 diffusion by means of a wastewater-based environmental monitoring developed in Piedmont, N-W Italy, during the second and third pandemic waves. Wastewater sampling strategies, sampling points sample pre-treatments and analytical methods, data processing and standardization, have been developed and discussed to give representative and reliable results. The following outcomes has been highlighted by the present study: i) a strong correlation between SARS-CoV-2 concentration in untreated wastewater and epidemic evolution in the considered areas can be observed as well as a predictive potential that could provide decision-makers with indications to implement effective policies, to mitigate the effects of the ongoing pandemic and to prepare response plans for future pandemics that could certainly arise in the decades to come; ii) moreover, the data at disposal from our monitoring campaign (almost 500 samples analysed in 11 months) confirm that SARS-CoV-2 concentrations in wastewater are strongly variable and site-specific across the region: the highest SARS-CoV-2 concentration values have been found in sewer networks serving the most populated areas of the region; iii) normalization of viral concentrations in wastewater through Pepper Mild Mottle Virus (a specific faecal marker) has been carried out and commented; iv) the study highlights the potential of wastewater treatment plants to degrade the genetic material referable to SARS-CoV-2 as well. In conclusion, the preliminary data reported in the present paper, although they need to be complemented by further studies considering also other geographical regions, are very promising., (Copyright © 2021 Elsevier Inc. All rights reserved.)

One of the main concerns in human milk banks (HMB) is the transmission of human cytomegalovirus (HCMV) that could be present in the milk of infected women. There are consistent data showing that this virus is destroyed by Holder pasteurization (62.5°C for 30 min), but there is a lack of information about the response of the virus to the treatment at lower temperatures in strict HMB conditions. In order to analyze the effectiveness of different temperatures of pasteurization to eliminate HCMV in human milk, a preliminary assay was performed incubating HCMV-spiked raw milk samples from donor mothers at tested temperatures in a PCR thermocycler and the viral infectivity was assayed on cell cultures. No signs of viral replication were observed after treatments at temperatures equal or >53°C for 30, 20, and 10 min, 58°C for 5 min, 59°C for 2 min, and 60°C for 1 min. These data were confirmed in a pasteurizer-like model introducing HCMV-spiked milk in disposable baby bottles. No viral infectivity was detected on cell cultures after heating treatment of milk for 30 min at temperatures from 56 to 60°C. Thus, our results show that by using conventional pasteurization conditions, temperatures in the range of 56-60°C are enough to inactivate HCMV. Consequently, we consider that, in order to provide a higher quality product, the current recommendation to pasteurize both mother's own milk and donated milk at 62.5°C must be re-evaluated., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gayà, Rittà, Lembo, Tonetto, Cresi, Sottemano, Bertino, Moro, Calvo and Donalisio.)

Since the beginning of coronavirus disease 2019 (COVID-19) pandemic, large attention has been focused on the relationship between SARS-CoV-2 diffusion and environment. As a matter of fact, clear evidence of the transmission of SARS-CoV-2 via respiratory aerosol would be of primary importance; at the same time, checking the presence of SARS-CoV-2 in wastewater can be extremely useful to control the diffusion of the disease. Up to now, many studies report SARS-CoV-2 concentrations in indoor/outdoor air samples or water/wastewater samples that can differ by order of magnitude. Unfortunately, complete information about the scientific approach of many studies is still missing, relating to: samplers and sampling materials performances, recovery tests, measurement uncertainty, robustness, detection and quantification limits, infectivity of captured virus, virus degradation during sampling, influence of sample pre-treatments (included freezing) on results, effects of inhibitors, sample alterations due to manipulation, validation of methods and processes, quality assurance according to ISO/IEC 17025 requirements. Based on the first experiences focused on the presence of SARS-CoV-2 in environmental samples such as air quality filters or impingers collection solutions, the present study describes a coherent preliminary approach to SARS-CoV-2 indoor and outdoor air sampling in order to overcome the evident lack of standardization. Three aspects are highlighted here: the first solution to assure quality and consistency to air sampling relies on the development of recovery tests using standard materials and investigating sampling materials, sampling techniques, sampling durations, sample conservation and pre-treatments; secondly, in order to overcome the shortcomings of every single sampling technique, coupling different samplers in parallel sampling could be an efficient strategy to collect more information and make data more reliable; finally, with regards to airborne virus sampling, the results could be confirmed by simplified emission and dilution models., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: It is known that breastfeeding protects the infant from enteric and respiratory infections; however, the antiviral properties of human milk against enteric and respiratory viruses are largely unexplored., Research Aims: To explore the antiviral activity of human preterm colostrum against rotavirus and respiratory syncytial virus and to assess whether the derived extracellular vesicle contribute to this activity., Methods: We used a cross-sectional, prospective two-group non-experimental design. Colostra were collected from mothers of preterm newborns ( N = 10) and extracellular vesicles were purified and characterized. The antiviral activity of colostra and derived extracellular vesicles were tested in vitro against rotavirus and respiratory syncytial virus and the step of viral replication inhibited by extracellular vesicles was investigated., Results: Each sample of colostrum and colostrum-derived extracellular vesicles had significant antiviral activity with a wide interpersonal variability. Mechanism of action studies demonstrated that extracellular vesicles acted by interfering with the early steps of the viral replicative cycle., Conclusion: We demonstrated the intrinsic antiviral activity of human colostrum against rotavirus and respiratory syncytial virus and we showed that extracellular vesicles substantially contribute to the overall protective effect. Our results contribute to unravelling novel mechanisms underlying the functional role of human milk as a protective and therapeutic agent in preterm infants.

Zika virus, an arthropod-borne flavivirus, is an emerging healthcare threat worldwide. Zika virus is responsible for severe neurological effects, such as paralytic Guillain-Barrè syndrome, in adults, and also congenital malformations, especially microcephaly. No specific antiviral drugs and vaccines are currently available, and treatments are palliative, but medicinal plants show great potential as natural sources of anti-Zika phytochemicals. This study deals with the investigation of the composition, cytotoxicity, and anti-Zika activity of Punica granatum leaf ethanolic extract, fractions, and phytoconstituents. P. granatum leaves were collected from different areas in Italy and Greece in different seasons. Crude extracts were analyzed and fractionated, and the pure compounds were isolated. The phytochemical and biomolecular fingerprint of the pomegranate leaves was determined. The antiviral activities of the leaf extract, fractions, and compounds were investigated against the MR766 and HPF2013 Zika virus strains in vitro . Both the extract and its fractions were found to be active against Zika virus infection. Of the compounds isolated, ellagic acid showed particular anti-Zika activities, with EC 50 values of 30.86 µM for MR766 and 46.23 µM for HPF2013. The mechanism of action was investigated using specific antiviral assays, and it was demonstrated that ellagic acid was primarily active as it prevented Zika virus infection and was able to significantly reduce Zika virus progeny production. Our data demonstrate the anti-Zika activity of pomegranate leaf extract and ellagic acid for the first time. These findings identify ellagic acid as a possible anti-Zika candidate compound that can be used for preventive and therapeutic interventions., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Background: In the current SARS-Coronavirus-2 (SARS-CoV-2) pandemic little is known about SARS-CoV-2 in human milk. It is important to discover if breast milk is a vehicle of infection. Objective: Our aim was to look for the presence of SARS-CoV-2 RNA in the milk of a group of SARS-CoV-2 positive mothers from North-West Italy. Methods: This is a prospective collaborative observational study where samples of human milk from 14 breastfeeding mothers positive for SARS-CoV-2 were collected. A search of viral RNA in breast milk samples was performed by RT-PCR (Real-Time reverse-transcriptase-Polymerase-Chain-Reaction) methodology tested for human milk. All the newborns underwent a clinical follow up during the first month of life or until the finding of two sequential negative swabs. Results: In 13 cases the search for SARS-CoV-2 RNA in milk samples resulted negative and in one case it was positive. Thirteen of the 14 newborns were exclusively breastfed and closely monitored in the first month of life. Clinical outcome was uneventful. Four newborns tested positive for SARS-CoV-2 and were all detected in the first 48 h of life, after the onset of maternal symptoms. Also the clinical course of these 4 infants, including the one who received mother's milk positive for SARS-CoV-2, was uneventful, and all of them became SARS-CoV-2 negative within 6 weeks of life. Conclusion: Our study supports the view that SARS-CoV-2 positive mothers do not expose their newborns to an additional risk of infection by breastfeeding., (Copyright © 2020 Bertino, Moro, De Renzi, Viberti, Cavallo, Coscia, Rubino, Tonetto, Sottemano, Campagnoli, Soldi, Mostert, Cresi, Lembo and Collaborative Research Group on SARS-CoV-2 in Human Milk.)

The benefits of human milk are mediated by multiple nutritional, trophic, and immunological components, able to promote infant's growth, maturation of its immature gut, and to confer protection against infections. Despite these widely recognized properties, breast-feeding represents an important mother-to-child transmission route of some viral infections. Different studies show that some flaviviruses can occasionally be detected in breast milk, but their transmission to the newborn is still controversial. The aim of this study is to investigate the antiviral activity of human milk (HM) in its different stages of maturation against two emerging flaviviruses, namely Zika virus (ZIKV) and Usutu virus (USUV) and to verify whether HM-derived extracellular vesicles (EVs) and glycosaminoglycans (GAGs) contribute to the milk protective effect. Colostrum, transitional and mature milk samples were collected from 39 healthy donors. The aqueous fractions were tested in vitro with specific antiviral assays and EVs and GAGs were derived and characterized. HM showed antiviral activity against ZIKV and USUV at all the stages of lactation with no significant differences in the activity of colostrum, transitional or mature milk. Mechanism of action studies demonstrated that colostrum does not inactivate viral particles, but it hampers the binding of both flaviviruses to cells. We also demonstrated that HM-EVs and HM-GAGs contribute, at least in part, to the anti-ZIKV and anti-USUV action of HM. This study discloses the intrinsic antiviral activity of HM against ZIKV and USUV and demonstrates the contribution of two bioactive components in mediating its protective effect. Since the potential infectivity of HM during ZIKV and USUV infection is still unclear, these data support the World Health Organization recommendations about breast-feeding during ZIKV infection and could contribute to producing new guidelines for a possible USUV epidemic., Competing Interests: The authors have declared that no competing interests exist.

There is an urgent need to identify antivirals against the coronavirus SARS-CoV-2 in the current COVID-19 pandemic and to contain future similar emergencies early on. Specific side-chain cholesterol oxidation products of the oxysterols family have been shown to inhibit a large variety of both enveloped and non-enveloped human viral pathogens. Here we report on the in vitro inhibitory activity of the redox active oxysterol 27-hydroxycholesterol against SARS-CoV-2 and against one of the common cold agents HCoV-OC43 human coronavirus without significant cytotoxicity. Interestingly, physiological serum levels of 27-hydroxycholesterol in SARS-CoV-2 positive subjects were significantly decreased compared to the matched control group, reaching a marked 50% reduction in severe COVID-19 cases. Moreover, no correlation at all was observed between 24-hydroxycholesterol and 25-hydroxycholesterol serum levels and the severity of the disease. Opposite to that of 27-hydroxycholesterol was the behaviour of two recognized markers of redox imbalance, i.e. 7-ketocholesterol and 7β-hydroxycholesterol, whose serum levels were significantly increased especially in severe COVID-19. The exogenous administration of 27-hydroxycholesterol may represent in the near future a valid antiviral strategy in the worsening of diseases caused by present and emerging coronaviruses., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Breast milk is a complex biofluid that nourishes infants, supports their growth and protects them from diseases. However, at the same time, breastfeeding is a transmission route for human cytomegalovirus (HCMV), with preterm infants being at a great risk of congenital disease. The discrepancy between high HCMV transmission rates and the few reported cases of infants with severe clinical illness is likely due to the protective effect of breast milk. The aim of this study was to investigate the anti-HCMV activity of human preterm colostrum and clarify the role of colostrum-derived extracellular vesicles (EVs). Preterm colostrum samples were collected and the EVs were purified and characterized. The in vitro anti-HCMV activity of both colostrum and EVs was tested against HCMV, and the viral replication step inhibited by colostrum-purified EVs was examined. We investigated the putative role EV surface proteins play in impairing HCMV infection using shaving experiments and proteomic analysis. The obtained results confirmed the antiviral action of colostrum against HCMV and demonstrated a remarkable antiviral activity of colostrum-derived EVs. Furthermore, we demonstrated that EVs impair the attachment of HCMV to cells, with EV surface proteins playing a role in mediating this action. These findings contribute to clarifying the mechanisms that underlie the protective role of human colostrum against HCMV infection.

We have synthesized and structurally characterized three tetra-( p -tolyl)antimony(III)-containing heteropolytungstates, [{( p -tolyl)Sb III } 4 ( A -α-XW 9 O 34 ) 2 ] n - [X = P V ( 1-P ), As V ( 1-As ), or Ge IV ( 1-Ge )], in aqueous solution using conventional, one-pot procedures. The polyanions 1-P , 1-As , and 1-Ge were fully characterized in the solid state and in solution and were shown to be soluble and stable in aqueous medium at pH 7. Biological studies demonstrated that all three polyanions possess significant antibacterial and antitumor activities. The minimum inhibitory concentrations of 1-P , 1-As , and 1-Ge were determined against four kinds of bacteria, including the two pathogenic bacteria strains, Vibrio parahaemolyticus and Vibrio vulnificus . The three novel polyanions also showed high cytotoxic potency in the human cell lines A549 (non-small cell lung cancer), CH1/PA-1 (ovarian teratocarcinoma), and SW480 (colon carcinoma).

Infections caused by HSV-2 are a public health concern worldwide, and there is still a great demand for the discovery of novel anti-herpes virus agents effective against strains resistant to current antiviral agents. In this context, medicinal plants represent an alternative source of active compounds for developing efficient antiviral therapies. The aim of this study was to evaluate the antiviral activity of Arisaema tortuosum , a plant used in the traditional medicine of India. A chloroform soluble fraction of the leaves exhibited anti-HSV-2 activity with a selectivity index of 758. The extract was also active against acyclovir-resistant HSV-2 and HSV-1. The mechanism of action of the extract was investigated evidencing inhibition of both early and late events of the HSV-2 replicative cycle. A HPLC-PDA-MS/MS analysis showed the presence of flavonoids including apigenin and luteolin in the chloroform extract (CE). Apigenin and luteolin showed a high inhibitory activity with EC 50 values of 0.05 and 0.41 µg/mL, respectively. Both compounds exhibited antiviral activity when added up to 6 h post infection and were able to reduce the viral progeny production. In addition, apigenin interfered with cell-to-cell virus spread., Competing Interests: The authors declare that they have no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

Physiological cholesterol metabolism implies the generation of a series of oxidized derivatives, whose oxysterols are by far the most investigated ones for their potential multifaceted involvement in human pathophysiology. In this regard, noteworthy is the broad antiviral activity displayed by defined side chain oxysterols, in particular 25-hydroxycholesterol (25HC) and 27-hydroxycholesterol (27HC). Although their antiviral mechanism(s) may vary depending on virus/host interaction, these oxysterols share the common feature to hamper viral replication by interacting with cellular proteins. Here reported is the first analysis of the modulation of a cell proteome by these two oxysterols, that, besides yielding additional clues about their potential involvement in the regulation of sterol metabolism, provides novelinsights about the mechanism underlying the inhibition of virus entry and trafficking within infected cells. We show here that both 25HC and 27HC can down-regulate the junction adhesion molecule-A (JAM-A) and the cation independent isoform of mannose-6-phosphate receptor (MPRci), two crucial molecules for the replication of all those viruses that exploit adhesion molecules and the endosomal pathway to enter and diffuse within target cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Human rotaviruses represent a major cause of severe diarrheal disease in infants and young children. The limited impact of oral vaccines on global estimates of rotavirus mortality and the suboptimal use of oral rehydration justify the need for alternative prophylactic and therapeutic strategies, especially for immunocompromised hosts. The protective effects of colostrum-the first milk produced during the initial 24 to 48 h after parturition-are well documented in the literature. In particular, the ingestion of hyperimmune bovine colostrum has been proposed as an alternative preventive approach against human rotavirus gastroenteritis. Although the immunization of pregnant cows with human rotavirus boosts the release of specific immunoglobulin G in bovine colostrum, it raises regulatory and safety issues. In this study, we demonstrated that the conventional bovine rotavirus vaccine is sufficient to enhance the anti-human rotavirus protective efficacy of bovine colostrum, thus providing a conservative approach to produce hyperimmune bovine colostrum, making it exploitable as a functional food., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

In the current study, fucoidans from brown alga Nizamuddinia zanardinii were isolated with conventional and non-conventional extraction procedures to evaluate the effects of recently introduced technologies on biochemical characteristics and saccharide composition of the extracts, along with their antibacterial, antiviral and cytotoxic properties. The results demonstrated that subcritical water extraction showed the highest fucoidans yield (13.15%), while the lowest yield was obtained using ultrasound extraction method (3.6%). The polysaccharide chains consisted of fucose, galactose, glucose, mannose and xylose, whose molar percentages differed according to the extraction method used. The weight mean average molecular weight of fucoidans varied between 444 and 1184 kDa. The FT-IR spectroscopy confirmed the presence of sulfate esters by bending vibration of COS and stretching vibration of SO peaks at 818 and 1250 cm -1 , respectively. Antibacterial assays showed that microwave- and subcritical water-extracted fucoidans inhibited the growth of E.coli and that enzyme-ultrasound, ultrasound-microwave and subcritical water extracted fucoidans exhibited inhibitory effects against P. aeruginosa at 2 mg/mL. Antiviral studies revealed that all the extracted fucoidans exerted strong antiviral activity against HSV-2 infection, with EC 50 values in the 0.027-0.123 μg/mL range; indeed the viscozyme-extracted macromolecules displayed the best selectivity index., (Copyright © 2018. Published by Elsevier B.V.)

Zika virus (ZIKV) is an emerging infectious viral pathogen associated with severe fetal cerebral anomalies and the paralytic Guillain-Barrè syndrome in adults. It was the cause of a recent global health crisis following its entrance into a naïve population in the Americas. Nowadays, no vaccine or specific antiviral against ZIKV is available. In this study, we identified three polyoxometalates (POMs), the Anderson-Evans type [TeW 6 O 24 ] 6- (TeW 6 ), and the Keggin-type [TiW 11 CoO 40 ] 8- &#95; (TiW 11 Co), and [Ti 2 PW 10 O 40 ] 7- (Ti 2 PW 10 ), that inhibit ZIKV infection with EC 50 s in the low micromolar range. Ti 2 PW 10 , the POM with the greatest selectivity index (SI), was selected and the step of ZIKV replicative cycle putatively inhibited was investigated by specific antiviral assays. We demonstrated that Ti 2 PW 10 targets the entry process of ZIKV infection and it is able to significantly reduce ZIKV progeny production. These results suggest that the polyanion Ti 2 PW 10 could be a good starting point to develop an effective therapeutic to treat ZIKV infection., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: A mother's own milk (MOM) is the gold standard for the feeding and nutrition of preterm and full term infants. When MOM is not available or there is not enough, donor human milk (DHM) should be used. Milk delivered to Human Milk Banks (HMBs) should be pasteurized to inactivate viral and bacterial agents. Currently, a pasteurization process at 62.5°C for 30 min (Holder pasteurization, HoP) is recommended in all international HMBs guidelines. State of the art: It is known that HoP affects some of the nutritional and biological components of human milk. Studies have demonstrated that temperature cycle in HoP is not always controlled or calibrated. A better check of these parameters in the pasteurizers on the market today may contribute to an improvement of the quality of HM, still maintaining some of the negative effects of the heat treatment of human milk. So, food industry, and dairy industry in particular, are evaluating innovative methodologies alternative to HoP to better preserve the nutritional and biological properties of fresh human milk, while assuring at least the same microbiological safety of HoP. The most studied processing techniques include High-Temperature-Short-Time (HTST) pasteurization, High Pressure Processing (HPP), and Ultraviolet-C (UV-C) irradiation. HTST is a thermal process in which milk is forced between plates or pipes that are heated on the outside by hot water at a temperature of 72°C for 5-15 s. HPP is a non-thermal processing method that can be applied to solid and liquid foods. This technology inactivates pathogenic microorganisms by applying a high hydrostatic pressure (usually 300-800 MPa) during short-term treatments (<5-10 min). UV irradiation utilizes short-wavelength ultraviolet radiation in the UV-C region (200-280 nm), which is harmful to microorganisms. It is effective in destroying the nucleic acids in these organisms, so that their DNA is disrupted by UV radiation. Aim: The aim of this paper is to present the EMBA recommendations on processing of HM, based on the most recent results obtained with these new technologies. Conclusions: Although research on the most promising technologies that will represent an alternative to HoP (HTST, HPP, UV-C) in the future is progressing, it is now important to recognize that the consistency and quality assurance of the pasteurizers on the market today represent a fundamental component that was previously lacking in the Holder approach.

Viral infections are an important cause of death worldwide. Unfortunately, there is still a lack of antiviral drugs or vaccines for a large number of viruses, and this represents a remarkable challenge particularly for emerging and re-emerging viruses. For this reason, the identification of broad spectrum antiviral compounds provides a valuable opportunity for developing efficient antiviral therapies. Here we report on a class of rhodanine and thiobarbituric derivatives displaying a broad spectrum antiviral activity against seven different enveloped viruses including an HSV-2 acyclovir resistant strain with favorable selectivity indexes. Due to their selective action on enveloped viruses and to their lipid oxidation ability, we hypothesize a mechanism on the viral envelope that affects the fluidity of the lipid bilayer, thus compromising the efficiency of virus-cell fusion and preventing viral entry., Competing Interests: The commercial affiliation to Lead Discovery Siena srl does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Objectives: This study aimed to investigate the anti-human cytomegalovirus (CMV) activity of milk from seropositive and seronegative mothers of preterm infants and to analyze its changes throughout the different stages of lactation and after Holder pasteurization, a procedure adopted by donor human milk banks., Methods: Eighteen mothers of preterm infants were enrolled in the study. Colostrum, transitional milk, and mature milk samples were collected and tested for anti-CMV activity. Depletion of immunoglobulins A from milk samples was carried out by jacalin resin. Pools of milk samples were pasteurized according to Holder technique., Results: All samples were endowed with anti-CMV activity, although to a different extent. In CMV IgG-positive mothers, colostra were significantly more active than the transitional milk and mature milk samples. Moreover, they were more potent than colostra from seronegative mothers. Immunoglobulins A depletion in colostra from IgG-positive mothers resulted in a partial loss of anti-CMV activity. Holder pasteurization significantly reduced the antiviral activity., Conclusions: Human milk is endowed with anti-CMV activity and its potency may vary depending on the stage of lactation and the serological status of the mother. This biological property could partially neutralize CMV particles excreted in the milk of CMV IgG-positive mothers thus reducing the risk of transmitting infectious viruses to the infant.

Holder pasteurization (62. 5°C for 30 min) is recommended by all international human milk bank guidelines to prevent infections potentially transmitted by donor human milk. A drawback is that it affects some human milk bioactive and nutritive components. Recently, High Temperature-Short Time (HTST) pasteurization has been reported to be a valuable alternative technology to increase the retention of some biological features of human milk. Nevertheless, to date, few data are available about the impact of pasteurization methods other than Holder on the antiviral activity of human milk. The present study was aimed at evaluating the antiviral activity of human milk against a panel of viral pathogens common in newborns and children (i.e., herpes simplex virus 1 and 2, cytomegalovirus, respiratory syncytial virus, rotavirus, and rhinovirus), and at assessing the effect of Holder and HTST pasteurization on milk's antiviral properties. The results indicate that human milk is endowed with antiviral activity against all viruses tested, although to a different extent. Unlike the Holder pasteurization, HTST preserved the inhibitory activity against cytomegalovirus, respiratory syncytial virus, rotavirus and herpes simplex virus type 2. By contrast, both methods reduced significantly the antiviral activities against rhinovirus and herpes simplex virus type 1. Unexpectedly, Holder pasteurization improved milk's anti-rotavirus activity. In conclusion, this study contributes to the definition of the pasteurization method that allows the best compromise between microbiological safety and biological quality of the donor human milk: HTST pasteurization preserved milk antiviral activity better than Holder.

A novel innate immune strategy, involving specific cholesterol oxidation products as effectors, has begun to reveal connections between cholesterol metabolism and immune response against viral infections. Indeed, 25-hydroxycholesterol (25HC) and 27-hydroxycholesterol (27HC), physiologically produced by enzymatic oxidation of cholesterol, act as inhibitors of a wide spectrum of enveloped and non-enveloped human viruses. However, the mechanisms underlying their protective effects against non-enveloped viruses are almost completely unexplored. To get insight into this field, we investigated the antiviral activity of 25HC and 27HC against a non-enveloped virus causing acute gastroenteritis in children, the human rotavirus (HRV). We found that 25HC and 27HC block the infectivity of several HRV strains at 50% inhibitory concentrations in the low micromolar range in the absence of cell toxicity. Both molecules affect the final step of virus penetration into cells by preventing the association of two cellular proteins: the oxysterol binding protein (OSBP) and the vesicle-associated membrane protein-associated protein-A (VAP-A). By altering the activity of these cellular mediators, 25HC and 27HC disturb the recycling of cholesterol between the endoplasmic reticulum and the late endosomes which are exploited by HRV to penetrate into the cell. The substantial accumulation of cholesterol in the late endosomal compartment results in sequestering viral particles inside these vesicles thereby preventing cytoplasmic virus replication. These findings suggest that cholesterol oxidation products of enzymatic origin might be primary effectors of host restriction strategies to counteract HRV infection and point to redox active lipids involvement in viral infections as a research area of focus to better focus in order to identify novel antiviral agents targets., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Although highly active antiretroviral therapies (HAART) remarkably increased life expectancy of HIV positive people, the rate of novel HIV-1 infections worldwide still represent a major concern. In this context, pre-exposure prophylaxis (PrEP) approaches such as vaginal microbicide gels topically releasing antiretroviral drugs, showed to have a striking impact in limiting HIV-1 spread. Nevertheless, the co-presence of other genital infections, particularly those due to HSV-1 or 2, constitute a serious drawback that strongly limits the efficacy of PrEP approaches. For this reason, combinations of different compounds with mixed antiviral and antiretroviral activity are thoroughly investigated Here we report the synthesis and the biological evaluation of a novel series of rhodanine derivatives, which showed to inhibit both HIV-1 and HSV-1/2 replication at nanomolar concentration, and were found to be active also on acyclovir resistant HSV-2 strains. The compounds showed a considerable reduction of activity in presence of serum due to a high binding to serum albumin, as determined through in vitro ADME evaluations. However, the most promising compound of the series maintained a considerable activity in gel formulation, with an EC50 comparable to that obtained for the reference drug tenofovir. Moreover, the series of compounds showed pharmacokinetic properties suitable for topical formulation, thus suggesting that the novel rhodanine derivatives could represent effective agents to be used as dual anti HIV/HSV microbicides in PrEP approaches., Competing Interests: The commercial affiliation to Lead Discovery Siena srl does not alter our adherence to PLOS ONE policies on sharing data and materials.

Viral infections kill millions yearly. Available antiviral drugs are virus-specific and active against a limited panel of human pathogens. There are broad-spectrum substances that prevent the first step of virus-cell interaction by mimicking heparan sulfate proteoglycans (HSPG), the highly conserved target of viral attachment ligands (VALs). The reversible binding mechanism prevents their use as a drug, because, upon dilution, the inhibition is lost. Known VALs are made of closely packed repeating units, but the aforementioned substances are able to bind only a few of them. We designed antiviral nanoparticles with long and flexible linkers mimicking HSPG, allowing for effective viral association with a binding that we simulate to be strong and multivalent to the VAL repeating units, generating forces (∼190 pN) that eventually lead to irreversible viral deformation. Virucidal assays, electron microscopy images, and molecular dynamics simulations support the proposed mechanism.  These particles show no cytotoxicity, and in vitro nanomolar irreversible activity against herpes simplex virus (HSV), human papilloma virus, respiratory syncytial virus (RSV), dengue and lenti virus. They are active ex vivo in human cervicovaginal histocultures infected by HSV-2 and in vivo in mice infected with RSV.

Ficus religiosa extracts have been used in traditional Indian medicine to treat sexually transmitted infections such as gonorrhea and genital ulcers. The aim of this study was to investigate the antiviral activity of F. religiosa extracts against herpes simplex virus type 2 (HSV-2), the main causative agent of genital ulcers and sores. Water and chloroform bark extracts were the most active against HSV-2, and also against an acyclovir-resistant strain. We demonstrate that the water extract has a direct virus-inactivating activity. By contrast, the chloroform extract inhibits viral attachment and entry and limits the production of viral progeny.

Up to 80% of the cost of vaccination programmes is due to the cold chain problem (that is, keeping vaccines cold). Inexpensive, biocompatible additives to slow down the degradation of virus particles would address the problem. Here we propose and characterize additives that, already at very low concentrations, improve the storage time of adenovirus type 5. Anionic gold nanoparticles (10 -8 -10 -6  M) or polyethylene glycol (PEG, molecular weight ∼8,000 Da, 10 -7 -10 -4  M) increase the half-life of a green fluorescent protein expressing adenovirus from ∼48 h to 21 days at 37 °C (from 7 to >30 days at room temperature). They replicate the known stabilizing effect of sucrose, but at several orders of magnitude lower concentrations. PEG and sucrose maintained immunogenicity in vivo for viruses stored for 10 days at 37 °C. To achieve rational design of viral-vaccine stabilizers, our approach is aided by simplified quantitative models based on a single rate-limiting step.

The development of topical microbicides is a valid approach to protect the genital mucosa from sexually transmitted infections that cannot be contained with effective vaccination, like HSV and HIV infections. A suitable target of microbicides is the interaction between viral proteins and cell surface heparan sulfate proteoglycans (HSPGs). AGMA1 is a prevailingly cationic agmatine-containing polyamidoamine polymer previously shown to inhibit HSPGs dependent viruses, including HSV-1, HSV-2, and HPV-16. The aim of this study was to elucidate the mechanism of action of AGMA1 against HSV infection and assess its antiviral efficacy and biocompatibility in preclinical models. The results show AGMA1 to be a non-toxic inhibitor of HSV infectivity in cell cultures and human cervicovaginal histocultures. Moreover, it significantly reduced the burden of infection of HSV-2 genital infection in mice. The investigation of the mechanism of action revealed that AGMA1 reduces cells susceptibility to virus infection by binding to cell surface HSPGs thereby preventing HSV attachment. This study indicates that AGMA1 is a promising candidate for the development of a topical microbicide to prevent sexually transmitted HSV infections., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Human papilloma virus (HPV)-16 is the prevalent genotype associated with cervical tumours. Virus-like-particle (VLP)-based vaccines have proven to be effective in limiting new infections of high-risk HPVs, but their high cost has hampered their use, especially in the poor developing countries. Avipox-based recombinants are replication-restricted to avian species and represent efficient and safe vectors also for immunocompromised hosts, as they can elicit a complete immune response. A new fowlpox virus recombinant encoding HPV-L1 (FPL1) was engineered and evaluated side-by-side with a FP recombinant co-expressing L1 and green fluorescent protein (FPL1GFP) for correct expression of L1 in vitro in different cell lines, as confirmed by Western blotting, immunofluorescence, real-time PCR, and electron microscopy. Mice were also immunised to determine its immunogenicity. Here, we demonstrate that the FPL1 recombinant better expresses L1 in the absence of GFP, correctly assembles structured capsomers into VLPs, and elicits an immune response in a preclinical animal model. To our knowledge, this is the first report of HPV VLPs assembled in eukaryotic cells using an avipox recombinant., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A new, simple and green method was developed for the manufacturing of heparin nanoassemblies active against the heparan sulfate-dependent viruses HSV-1, HSV-2, HPV-16 and RSV. These nanoassemblies were obtained by the auto-association of O-palmitoyl-heparin and α-cyclodextrin in water. The synthesized O-palmitoyl-heparin derivatives mixed with α-cyclodextrin resulted in the formation of crystalline hexagonal nanoassemblies as observed by transmission electron microscopy. The nanoassembly mean hydrodynamic diameters were modulated from 340 to 659 nm depending on the type and the initial concentration of O-palmitoyl-heparin or α-cyclodextrin. The antiviral activity of the nanoassemblies was not affected by the concentration of the components. However, the method of the synthesis of O-palmitoyl-heparin affected the antiviral activity of the formulations. We showed that reduced antiviral activity is correlated with lower sulfation degree and anticoagulant activity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Micro- and nanobubbles provide a promising non-viral strategy for ultrasound mediated gene delivery. Microbubbles are spherical gas-filled structures with a mean diameter of 1-8 μm, characterised by their core-shell composition and their ability to circulate in the bloodstream following intravenous injection. They undergo volumetric oscillations or acoustic cavitation when insonified by ultrasound and, most importantly, they are able to resonate at diagnostic frequencies. It is due to this behaviour that microbubbles are currently being used as ultrasound contrast agents, but their use in therapeutics is still under investigation. For example, microbubbles could play a role in enhancing gene delivery to cells: when combined with clinical ultrasound exposure, microbubbles are able to favour gene entry into cells by cavitation. Two different delivery strategies have been used to date: DNA can be co-administered with the microbubbles (i.e. the contrast agent) or 'loaded' in purposed-built bubble systems - indeed a number of different technological approaches have been proposed to associate genes within microbubble structures. Nanobubbles, bubbles with sizes in the nanometre order of magnitude, have also been developed with the aim of obtaining more efficient gene delivery systems. Their small sizes allow the possibility of extravasation from blood vessels into the surrounding tissues and ultrasound-targeted site-specific release with minimal invasiveness. In contrast, microbubbles, due to their larger sizes, are unable to extravasate, thus and their targeting capacity is limited to specific antigens present within the vascular lumen. This review provides an overview of the use of microbubbles as gene delivery systems, with a specific focus on recent research into the development of nanosystems. In particular, ultrasound delivery mechanisms, formulation parameters, gene-loading approaches and the advantages of nanometric systems will be described., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cyclodextrin-based nanosponges (NS) are solid nanoparticles, obtained from the cross-linking of cyclodextrins that have been proposed as delivery systems for many types of drugs. Various NS derivatives are currently under investigation in order that their properties might be tuned for different applications. In this work, new carboxylated cyclodextrin-based nanosponges (Carb-NS) carrying carboxylic groups within their structure were purposely designed as novel Acyclovir carriers. TEM measurements revealed their spherical shape and size of about 400 nm. The behaviour of Carb-NS, with respect to the incorporation and delivery of Acyclovir, was compared to that of NS, previously investigated as a drug carrier. DSC, XRPD and FTIR analyses were used to investigate the two NS formulations. The results confirm the incorporation of the drug into the NS structure and NS-Acyclovir interactions. The Acyclovir loading into Carb-NS was higher than that obtained using NS, reaching about 70% (w/w). In vitro release studies showed the release kinetics of Acyclovir from Carb-NS to be prolonged in comparison with those observed with NS, with no initial burst effect. The NS uptake into cells was evaluated using fluorescent Carb-NS and revealed the nanoparticle internalisation. Enhanced antiviral activity against a clinical isolate of HSV-1 was obtained using Acyclovir loaded in Carb-NS., (Copyright © 2012 Elsevier B.V. All rights reserved.)