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Background The side chain 27-hydroxycholesterol has been reported to inhibit the replication of several pathogen viruses, including herpes simplex virus, rhinovirus, rotavirus and SARS-CoV-2, in in vitro and ex vivo models. Objective In view of a future potential therapeutic use of 27-hydroxycholesterol, a pilot pharmacokinetic study was set up. Methods This active substance was complexed with 2-hydroxypropyl-β-cyclodextrin and orally administered in a single dose to CD1 male mice; its recovery in plasma and a few tissues up to 24 h post-treatment was evaluated. Results The absorption of the oxysterol by the small intestine was moderate, due to its physicochemical properties, but still relevant and rapid, showing a peak at 1 h after supplementation and being almost completed 24 h after treatment. 27-Hydroxycholesterol appeared to be a high hepatic extraction drug, possibly with an extrahepatic component contributing to the total clearance. Conclusions Following the oral 25 mg/kg dosing, plasma levels of 27-hydroxycholesterol showed an average steady-state concentration similar to that shown to be able to inhibit the replication of all viruses tested so far in in vitro models. Significance statement The first pharmacokinetic data relative to a natural oxysterol administered p.o. are reported. Data should contribute to further elucidate oxysterol pathophysiology and guide non-clinical studies aiming at investigating possible therapeutic use of 27-hydroxycholesterol or its analogs.

Introduction: The bacterial genotoxin colibactin is enriched in colorectal cancer (CRC) and promotes the accumulation of mutations that drive tumorigenesis. However, systematic assessment of its impact on DNA damage response is lacking and the effect of colibactin exposure on response to other genotoxic agents (such as chemotherapy) is missing. Materials and Methods: We implemented an in vitro bacteria-coculture system to assess the effect of colibactin on a representative subset of 40 molecularly and pharmacologically annotated CRC cell lines and in a panel of isogenic DDR KO cell lines we generated. We further validated our results in patient-derived organoids. Finally, we recapitulated prolonged exposure to colibactin occurring during tumorigenesis by chronically infecting sensitive cells until the emergence of a tolerant phenotype.} Results: We found that different cell lines display specific sensitivity to colibactin’s genotoxic stress: while colibactin-tolerant cells are capable to quickly and efficiently repair colibactin-induced DNA damage, sensitive cells lack this ability. Moreover, we found that homologous recombination (HR) proficiency discriminates colibactin-tolerant cells, which display higher levels of RAD51 foci (as marker of activation of HR) compared to sensitive cells upon infection with colibactin. Screening of isogenic DDR KO cell lines revealed that genetic inactivation of the intertwined pathways of HR (through KO of ATM) and replication stress (RS) response (through KO of ATRIP) significantly sensitized cells to colibactin. In addition, we found that restoration of HR activity was sufficient to induce a colibactin-tolerant phenotype in initially sensitive cell lines. Notably, thanks to a previous effort of pharmacological characterization of CRC cell lines in our lab, we found a significant correlation between sensitivity to colibactin and irinotecan active metabolite SN38, but not oxaliplatin. We validated the same correlation in patient-derived organoids annotated for response to SN38. While colibactin, SN38 and oxaliplatin all induced RS in treated cells, we found that colibactin and SN38 showed a similar DNA damage response which involved activation of ATM. Finally, chronic re-infection of sensitive, HR-deficient CRC cells with colibactin selected a tolerant phenotype characterized by restoration of HR activity. Of translational relevance, colibactin-tolerant derivative cells acquired cross-resistance to SN38 and PARP inhibitor olaparib but not to oxaliplatin. Conclusion: Our results shed novel insight into colibactin’s genotoxic mechanism and support a model in which colibactin both promotes tumorigenesis and acts as an evolutionary bottleneck which selects HR proficient CRC cells. Furthermore, our study provides preclinical evidence on colibactin’s role in promoting chemoresistance in colorectal cancer. Citation Format: Alberto Sogari, Emanuele Rovera, Nicole Megan Reilly, Simona Lamba, Mariangela Russo, Annalisa Lorenzato, Erika Durinikova, Livio Trusolino, Sabrina Arena, Manuela Donalisio, Federica Di Nicolantonio, David Lembo, Alberto Bardelli. Tolerance to colibactin correlates with response to chemotherapeutic agents in colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5900.

We have synthesized and structurally characterized three tetra-(p-tolyl)antimony(III)-containing heteropolytungstates, [{(p-tolyl)SbIII}4(A-α-XW9O34)2]n- [X = PV (1-P), AsV (1-As), or GeIV (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 EC50 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.

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.

Herpesviruses are highly prevalent in the human population, and frequent reactivations occur throughout life. Despite antiviral drugs against herpetic infections, the increasing appearance of drug-resistant viral strains and their adverse effects prompt the research of novel antiherpetic drugs for treating lesions. Peptides obtained from natural sources have recently become of particular interest for antiviral therapy applications. In this work, we investigated the antiviral activity of the peptide A-3302-B, isolated from a marine bacterium, Micromonospora sp., strain MAG 9-7, against herpes simplex virus type 1, type 2, and human cytomegalovirus. Results showed that the peptide exerted a specific inhibitory activity against HSV-2 with an EC50 value of 14 µM. Specific antiviral assays were performed to investigate the mechanism of action of A-3302-B. We demonstrated that the peptide did not affect the expression of viral proteins, but it inhibited the late events of the HSV-2 replicative cycle. In detail, it reduced the cell-to-cell virus spread and the transmission of the extracellular free virus by preventing the egress of HSV-2 progeny from the infected cells. The dual antiviral and previously reported anti-inflammatory activities of A-3302-B, and its effect against an acyclovir-resistant HSV-2 strain are attractive features for developing a therapeutic to reduce the transmission of HSV-2 infections., International Journal of Molecular Sciences, 23 (2), ISSN:1422-0067

Herpesviruses are highly prevalent in the human population, and frequent reactivations occur throughout life. Despite antiviral drugs against herpetic infections, the increasing appearance of drug-resistant viral strains and their adverse effects prompt the research of novel antiherpetic drugs for treating lesions. Peptides obtained from natural sources have recently become of particular interest for antiviral therapy applications. In this work, we investigated the antiviral activity of the peptide A-3302-B, isolated from a marine bacterium, Micromonospora sp., strain MAG 9-7, against herpes simplex virus type 1, type 2, and human cytomegalovirus. Results showed that the peptide exerted a specific inhibitory activity against HSV-2 with an EC50 value of 14 μM. Specific antiviral assays were performed to investigate the mechanism of action of A-3302-B. We demonstrated that the peptide did not affect the expression of viral proteins, but it inhibited the late events of the HSV-2 replicative cycle. In detail, it reduced the cell-to-cell virus spread and the transmission of the extracellular free virus by preventing the egress of HSV-2 progeny from the infected cells. The dual antiviral and previously reported anti-inflammatory activities of A-3302-B, and its effect against an acyclovir-resistant HSV-2 strain are attractive features for developing a therapeutic to reduce the transmission of HSV-2 infections.

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.

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_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_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_PMDA and the acyclovir complexation potential of βCD are attractive features for developing new therapeutic topical options against genital HSV-2 infection.

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.

Hepatitis B virus (HBV) infection is a global health problem with different immunological phases and therapeutic approaches. The serological condition of inactive carrier (IC) was recently well defined as a clinical and virological stable status, in which specific treatment is usually deferred, while the active chronic hepatitis B (CHB) condition requires an immediate treatment strategy. Recently, a possible broad antiviral effect of oxysterols, in particular 25-hydroxycholesterol (25OHC) and 27-hydroxycholesterol (27OHC), was observed, as most likely linked to the positive modulation of innate immunity, but no clear evidence is available about their possible role in chronic HBV infection. Thus, we examined the relationship between the plasma levels of oxysterols and the disease condition of 40 HBV patients, without treatment at the start of the study. Of these, 33 were ICs and 7 were active CHB subjects. A marked reduction of 25OHC and 27OHC plasma levels was detectable in all active CHB recruited patients, while the plasma values observed in ICs all remained within the physiological range. No difference was observed between the two groups of patients with regard to the plasma levels of 24-hydroxycholesterol (24OHC). Further, the plasma level of 27OHC ≥ 140 μg/L was shown to be predictive of an inactive carrier status. This cohort study points to 27OHC as a good candidate biomarker to differentiate active and inactive CHB status. An increasing bulk of research reports is supporting the very likely contribution of this oxysterol to the immunological control of chronic hepatitis B.

The most recent scientific studies have finally identified the airborne transmission of SARS-CoV-2 as significant. Therefore, the airborne transmission path for SARS-CoV-2 is of primary scientific and health-related interest as it could actually involve management, accessibility, use and functionality of many activities, including hospitals (where COVID wards represent only a part of the critical issues), schools, workplaces, offices, factories, means of transport, sports venues, and the outdoor environment. It is necessary to develop a sampling and analytical method for virus-laden bioaerosol that could be considered reliable and validated according to ISO/IEC 17025 requirements.The present paper defines samples pretreatments aiming at recover SARS-CoV-2 from glass-fiber and PTFE filters used by low and high-volume air samplers. Recovery test results focused on the sample concentration step carried out by means of ultracentrifugation are reported as well. Human coronavirus strain OC43 (a surrogate β-coronavirus with same SARS-CoV-2 particle structure) was used to validate each step of the recovery tests.We obtained the following results:-the recovery efficiency from glass-fiber filters and quartz filters could be strongly enhanced by using an elution buffer containing up to 40% of fetal calf serum.-the recovery efficiency of coronavirus OC43 (HCoV-OC43) from PTFE filters is much higher and easier than from glass-fiber filters; for glass-fiber filters, we found that a two-step procedure is necessary to elute viral infective particles: a 3 hour-shaking step, followed by a 30 seconds-vortexing step. For PTFE 60 minutes-shaking is enough.-the effect of suction time on filters could be resumed as follows: concerning 10cm glass-fiber filters, sampling durations up to 20 minutes at a flow rate of 500 L per minute do not affect recovery efficiencies. On the contrary, the recovery efficiency of infectious virions from 4.7cm PTFE filters decreases of a factor 2 after 3 hours of sampling at a flow rate of 20 L per minute.-the recovery efficiency of ultracentrifugation is 57%.Furthermore, the effect of the storage temperature of the filters immersed in the transport medium on the infectivity of HCoV-OC43 has been assessed.Based on the sampling techniques and the analytical methods developed as described in the present study, many field tests were carried out reporting virus concentrations up to 50 genomic copies per cubic meter of air in domestic environment with poor ventilation condition, whereas in hospital wards the detectable concentrations of SARS-CoV-2 were generally lower than 10 genomic copies per cubic meter of air.The developed methods, aiming at providing the community with reliable determinations about the presence of SARS-CoV-2 and other airborne pathogens in air, prove essential for the development, during the pandemic, of a coherent management of places (especially of crowded ones) such as means of transport, stations, gyms, theaters, cinemas.

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.

The current emergency of the novel coronavirus SARS-CoV2 urged the need for broad-spectrum antiviral drugs as the first line of treatment. Coronaviruses are a large family of viruses that already challenged humanity in at least two other previous outbreaks and are likely to be a constant threat for the future. In this work we developed a pipeline based on in silico docking of known drugs on SARS-CoV1/2 RNA-dependent RNA polymerase combined with in vitro antiviral assays on both SARS-CoV2 and the common cold human coronavirus HCoV-OC43. Results showed that certain drugs displayed activity for both viruses at a similar inhibitory concentration, while others were specific. In particular, the antipsychotic drug lurasidone and the antiviral drug elbasvir showed promising activity in the low micromolar range against both viruses with good selectivity index.

Oxysterols are products of enzymatic and/or chemical cholesterol oxidation. While some of the former possess broad antiviral activities, the latter mostly originate from the deterioration of the nutritional value of foodstuff after exposure to heat, light, radiation and oxygen, raising questions about their potential health risks. We evaluated the presence of selected oxysterols in bovine colostrum and monitored the evolution of their cholesterol ratio throughout an entire industrial-scale milk production chain and after industrially employed storage procedures of milk powders. We report here for the first time the presence of high levels of the enzymatic oxysterol 27-hydroxycholesterol (27OHC) in concentrations of antiviral interest in bovine colostrum (87.04 ng mL-1) that decreased during the first postpartum days (56.35 ng mL-1). Of note, this oxysterol is also observed in milk and milk products and is not negatively affected by industrial processing or storage. We further highlight an exponential increase of the non-enzymatic oxysterols 7β-hydroxycholesterol (7βOHC) and 7-ketocholesterol (7KC) in both whole (WMPs) and skimmed milk powders (SMPs) during prolonged storage, confirming their role as reliable biomarkers of cholesterol oxidation over time: after 12 months, 7βOHC reached in both SMPs and WMPs amounts that have been found to be potentially toxic in vitro (265.46 ng g-1 and 569.83 ng g-1, respectively). Interestingly, industrial processes appeared to affect the generation of 7βOHC and 7KC differently, depending on the presence of fat in the product: while their ratios increased significantly after skimming and processing of skimmed milk and milk products, this was not observed after processing whole milk and milk cream.

The current emergency of the novel coronavirus SARS-CoV-2 urged the need for broad-spectrum antiviral drugs as the first line of treatment. Coronaviruses are a large family of viruses that already challenged humanity in at least two other previous outbreaks and are likely to be a constant threat for the future. In this work we developed a pipeline based on in silico docking of known drugs on SARS-CoV RNA-dependent RNA polymerase combined with in vitro antiviral assays on both SARS-CoV-2 and the common cold human coronavirus HCoV-OC43. Results showed that certain drugs displayed activity for both viruses at a similar inhibitory concentration, while others were specific. In particular, the antipsychotic drug lurasidone and the antiviral drug elbasvir showed promising activity in the low micromolar range against both viruses with good selective index.

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, air-liquid impingers and wastewater samples, the present study describes a coherent preliminary approach to SARS-CoV-2 environmental sampling in order to overcome the evident lack of standardization. Three aspects are highlighted here: the first solution to assure quality and consistency to environmental 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, in particular for air samples; finally, with regards to airborne virus sampling, the results could be confirmed by simplified emission and dilution models.

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., Author summary ZIKV and USUV are emerging flaviviruses that cause conditions ranging from mild febrile diseases to more sever outcomes. ZIKV is associated with microcephaly in newborns and USUV neurotropism represents a growing concern for human health. We studied these viruses in the context of breast-feeding. Breast-milk is a complex biofluid to nourish infants, support their growth and to protect them from numerous diseases, but it also represents a transmission route of several infections. It has been reported that flaviviruses can occasionally be detected in breast-milk, with limited information existing about their possible transmission through breast-feeding. We therefore explored the intrinsic protective role of human milk against ZIKV and USUV infections in vitro and we also assessed the contribution of specific components in mediating this activity. We demonstrated that human milk is endowed with anti-ZIKV and anti-USUV activity at all maturation stages and that it acts by altering virus attachment to the host cell. This activity is mostly due to non-specific bioactive factors, including extracellular vesicles and glycosaminoglycans. Our findings support the use of fresh milk (or from donor banks) as the food of choice for nutrition and protection of newborns in a possible context of ZIKV or USUV epidemics.

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 EC50 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.

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., Graphical abstract Image 1, Highlights • 27-hydroxycholesterol (27OHC) inhibits the replication of SARS-CoV-2 by interfering with its entry into target cells. • The broad antiviral effect of 27OHC is also exerted against another β-coronavirus, HCoV-OC43. • Blood levels of 27OHC were decreased in SARS-CoV-2 infected individuals, especially in patients with severe COVID-19. • COVID-19 patients showed increased serum levels of 7-ketocholesterol and 7β-hydroxycholesterol, markers of oxidative stress.

We have synthesized and structurally characterized three tetra-(

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., Graphical abstract Image 1, Highlights • 25HC and 27HC, two physiological products of enzymatic cholesterol oxidation, modulate the proteome of HeLa cells, standard model system. • Cell proteome analysis was afforded by coupling mass spectrometry to stable isotope labelling by amino acids in cell culture (SILAC) technology. • The large majority of proteins significantly modulated by both 25HC and 27HC is related to sterol synthesis and metabolism. • Down-regulation of JAM-A and MPRci likely contributes to the broad antiviral activity of 25HC and 27HC.

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.

Background: In the current COVID-19 pandemic caused by the SARS-Coronavirus-2 (SARS-CoV-2) it is important to understand if breast milk may be a vehicle of infection. The possible transmission of the virus via breastfeeding is still largely unexplored. Methods: This is a prospective collaborative observational study where samples of human milk from 12 breastfeeding mothers positive for SARS-Coronavirus 2 were collected. Search for the viral RNA in breast milk samples was performed by real-time PCR methodology. All the newborns underwent a clinical follow up for the first month of life or until the finding of two sequential negative swabs. Findings: In eleven cases the search for SARS-CoV-2 RNA in milk samples resulted negative and in one case it was positive. Eleven of the twelve newborns were exclusively breastfed in the first month of life and closely monitored. Clinical outcome was uneventful. Four newborns tested positive for SARS-CoV-2 and were all detected in the first 48 hours 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. Interpretations: Our study supports the view that SARS-CoV-2 positive mothers do not expose their newborns to additional risk of infection by breastfeeding. This is an important message that should be given to mothers and healthcare providers in this moment of uncertainty due to the COVID-19 pandemic. Clearly, the recommended hygiene measures for direct breastfeeding, as well as for milk expression when a mother and her infant need to be separated, must be carefully followed. Funding: This Study was supported by University of Turin (grant RILO2019 to EB and DL). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The study was approved by the local Ethical Committee (protocol number 0039684), and informed consent to participate in the study was obtained from each mother.