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Roberta Cavalli,1 Agnese Bisazza,1 Michele Trotta,1 Monica Argenziano,1 Andrea Civra,2 Manuela Donalisio,2 David Lembo21Department of Pharmaceutical Sciences and Technology; 2Department of Clinical and Biological Sciences University of Turin, Turin, ItalyBackground: The development of nonviral gene delivery systems is one of the most intriguing topics in nanomedicine. However, despite the advances made in recent years, several key issues remain unsettled. One of the main problems relates to the difficulty in designing nanodevices for targeted delivery of genes and other drugs to specific anatomic sites. In this study, we describe the development of a novel chitosan nanobubble-based gene delivery system for ultrasound-triggered release.Methods and results: Chitosan was selected for the nanobubble shell because of its low toxicity, low immunogenicity, and excellent biocompatibility, while the core consisted of perfluoropentane. DNA-loaded chitosan nanobubbles were formed with a mean diameter of less than 300 nm and a positive surface charge. Transmission electron microscopic analysis confirmed composition of the core-shell structure. The ability of the chitosan nanobubbles to complex with and protect DNA was confirmed by agarose gel assay. Chitosan nanobubbles were found to be stable following insonation (2.5 MHz) for up to 3 minutes at 37°C. DNA release was evaluated in vitro in both the presence and absence of ultrasound. The release of chitosan nanobubble-bound plasmid DNA occurred after just one minute of insonation. In vitro transfection experiments were performed by exposing adherent COS7 cells to ultrasound in the presence of different concentrations of plasmid DNA-loaded nanobubbles. In the absence of ultrasound, nanobubbles failed to trigger transfection at all concentrations tested. In contrast, 30 seconds of ultrasound promoted a moderate degree of transfection. Cell viability experiments demonstrated that neither ultrasound nor the nanobubbles affected cell viability under these experimental conditions.Conclusion: Based on these results, chitosan nanobubbles have the potential to be promising tools for ultrasound-mediated DNA delivery.Keywords: chitosan, nanobubbles, transfection, DNA, gene carrier, ultrasound

Estimou-se a proporção de incidência de acidentes do trabalho na Cidade de Botucatu, São Paulo, Brasil, segundo sexo, idade, existência de contrato de trabalho e ocupações. Para tanto, foi realizado inquérito domiciliar em amostra aleatória sistemática por conglomerados (195 setores censitários). Coletaram-se informações de todos os moradores dos domicílios amostrados com idade maior que nove anos. Para aqueles que trabalhavam nos últimos noventa dias que precederam a entrevista, foram obtidas informações sobre ocupação, posição na ocupação, contrato de trabalho e ocorrência de acidentes. Foram estudados 9.626 domicílios residenciais (fração amostral de 0,26). A proporção de acidentes não-fatais no município foi de 3,3% (IC95%: 2,7-3,9), sendo maior em homens 4,5% (IC95%: 3,6-5,5), variando com o tipo de contrato de trabalho e grande grupo de ocupação. Comparando-se esses resultados com períodos anteriores, observa-se diminuição significativa do risco de acidentes na cidade.

INTRODUÇÃO: A maior expectativa de vida no País e a queda da mortalidade trazem desafios aos programas sociais de atenção ao idoso, em especial na área na saúde. O objetivo do estudo foi analisar a tendência da mortalidade por doenças do trato respiratório em idosos. MÉTODOS: O estudo foi realizado com dados de mortalidade em idosos no Estado de São Paulo, no período de 1980 a 1998. Os dados foram obtidos do Sistema de Informações de Mortalidade do Ministério da Saúde (SIM/MS/DATASUS). Trata-se de um estudo ecológico de séries temporais onde foram analisadas as tendências das taxas padronizadas de mortalidade por doenças do aparelho respiratório, segundo faixas etárias (60 a 69, 70 a 79 e 80 ou mais anos), utilizando modelos de regressão linear. Descreve-se a mortalidade proporcional das doenças respiratórias frente às demais causas. RESULTADOS: Observou-se, no período, tendência crescente das taxas de mortalidade para ambos os sexos, principalmente a partir da faixa etária de 80 anos ou mais, havendo aumento na proporção de óbitos por doenças respiratórias em relação às demais causas. CONCLUSÕES: As doenças respiratórias são causas preocupantes de hospitalização e morte, particularmente na população idosa. As ações de prevenção e assistência a estas causas, bem como maior investigação etiológica, devem ser priorizadas no atual contexto epidemiológico da saúde do idoso no Brasil.

Os homicídios tornaram-se um grave problema social no Brasil nas últimas duas décadas, com repercussões em diversas áreas. O objetivo deste estudo é representar por imagens tridimensionais a evolução dos homicídios no sexo masculino, durante os últimos vinte anos, na Região Metropolitana de São Paulo. São apresentados o número absoluto e a proporção de incidência de óbitos masculinos causados por homicídios, bem como o logaritmo natural do risco relativo masculino/feminino de óbito por homicídio, na Região Metropolitana de São Paulo, entre 1979 e 1998. A proporção de incidência de óbitos por homicídios no sexo masculino cresceu de 35 para 121 (x 10-5) na região e período estudados. São apresentadas sete figuras tridimensionais que, em seu conjunto, situam a evolução deste fenômeno de acordo com faixa etária, ano calendário e sexo. As figuras apresentadas permitem facilmente visualizar as quatro principais características dos homicídios entre a população masculina: nítida predominância entre adolescente e adultos jovens; aumento consistente e acentuado ano a ano; espraiamento tanto para as menores idades (abaixo dos 18) quanto para as maiores (acima dos trinta); e manutenção de incidência elevada nas idades superiores a quarenta anos no final do período estudado.

Trata-se de um inquérito amostral (502 entrevistas) realizado na região de Campinas SP, em Santa Bárbara D'Oeste (170.000 habitantes), por ser o primeiro município a registrar casos de dengue autóctone na região, desde 1995. Avaliou-se o conhecimento da população sobre o dengue, seu vetor e prevenção em 3 bairros da cidade. Estas informações foram comparadas com a presença de criadouros no ambiente domiciliar, em áreas com e sem transmissão. O bairro com melhores condições sociais e urbanas apresentaram conhecimento mais adequado sobre a doença, embora os bairros periféricos tenham sido priorizados em atividades educativas devido à ocorrência de casos. Observou-se criadouros em todas as áreas examinadas, em quantidades semelhantes. Constatou-se a distância entre conhecimento e mudanças de comportamento. Identificou-se as fontes de informações mais referidas e os criadouros predominantes nos domicílios. Os resultados deste inquérito podem servir como subsídios para (re)orientar ações educativas das equipes de controle de vetores, bem como avaliar um instrumento simplificado para acompanhamento do impacto do programa local de controle do dengue.

OBJETIVO: Analisar a tendência da letalidade e da incidência da doença meningocócica no período de 1993 a 1998 na região de Campinas, SP, abrangendo cinco municípios de seu entorno (1,2 milhões de habitantes). MÉTODOS: Foi realizado estudo longitudinal retrospectivo de todos os casos notificados (375) da doença meningocócica pela vigilância epidemiológica regional. Por meio de análise de regressão logística foram identificados os fatores associados ao aumento da letalidade dessa doença. RESULTADOS: Os anos de 1996 e de 1997 apresentaram maiores coeficientes de letalidade (23,8%), coincidindo com picos de incidência do sorogrupo B, altos percentuais de meningococcemia e menor investigação etiológica. Observou-se padrão sazonal e predomínio da circulação da Neisseria meningitidis das cepas B:4:P1.15 e C:2b:P1.3. Os fatores relacionados com o aumento da letalidade pela análise de regressão logística foram: presença de meningococcemia, com ou sem meningite (odds ratio ajustado (ORaj) 13,88 e intervalo de confiança de 95% (IC) 4,68-42,13); idade acima de 30 anos (ORaj 6,42; IC 2,32-17,80); idade inferior a 1 ano (ORaj 2,95; IC 1,55-5,63); e sorogrupo B (ORaj 2,33; IC 1,14- 4,79). CONCLUSÕES: A septicemia, a idade e o sorogrupo mostraram-se variáveis preditoras de morte. Em alguns anos os coeficientes de letalidade apresentaram-se altos, indicando a necessidade de investigação da qualidade e da agilidade da assistência à saúde na prevenção dos óbitos. O percentual de identificação etiológica dos casos dificultou conclusões mais precisas sobre o comportamento epidemiológico das cepas.

Abstract Background Many factors have been associated with circulation of the dengue fever virus and vector, although the dynamics of transmission are not yet fully understood. The aim of this work is to estimate the spatial distribution of the risk of dengue fever in an area of continuous dengue occurrence. Methods This is a spatial population-based case-control study that analyzed 538 cases and 727 controls in one district of the municipality of Campinas, São Paulo, Brazil, from 2006-2007, considering socio-demographic, ecological, case severity, and household infestation variables. Information was collected by in-home interviews and inspection of living conditions in and around the homes studied. Cases were classified as mild or severe according to clinical data, and they were compared with controls through a multinomial logistic model. A generalized additive model was used in order to include space in a non-parametric fashion with cubic smoothing splines. Results Variables associated with increased incidence of all dengue cases in the multiple binomial regression model were: higher larval density (odds ratio (OR) = 2.3 (95%CI: 2.0-2.7)), reports of mosquito bites during the day (OR = 1.8 (95%CI: 1.4-2.4)), the practice of water storage at home (OR = 2.5 (95%CI: 1.4, 4.3)), low frequency of garbage collection (OR = 2.6 (95%CI: 1.6-4.5)) and lack of basic sanitation (OR = 2.9 (95%CI: 1.8-4.9)). Staying at home during the day was protective against the disease (OR = 0.5 (95%CI: 0.3-0.6)). When cases were analyzed by categories (mild and severe) in the multinomial model, age and number of breeding sites more than 10 were significant only for the occurrence of severe cases (OR = 0.97, (95%CI: 0.96-0.99) and OR = 2.1 (95%CI: 1.2-3.5), respectively. Spatial distribution of risks of mild and severe dengue fever differed from each other in the 2006/2007 epidemic, in the study area. Conclusions Age and presence of more than 10 breeding sites were significant only for severe cases. Other predictors of mild and severe cases were similar in the multiple models. The analyses of multinomial models and spatial distribution maps of dengue fever probabilities suggest an area-specific epidemic with varying clinical and demographic characteristics.

To evaluate the prevalence of Mycobacterium leprae and Mycobacterium lepromatosis in road killed armadillos identified along Brazilian regions, samples of liver, spleen, muscle, ear, nose and tail were collected on highways from 78 animals. The armadillos were of four different species, Cabassous tatouay, Dasypus novemcinctus, Dasypus septemcinctus and Euphractus sexcinctus. After DNA extraction from two tissues, specific primers were used for the detection of each pathogen using SYBR green qualitative Real-Time PCR, and amplicons were sequenced. The species with the highest prevalence was D. novemcinctus, mainly in the Central-West, South, and Southeast regions of Brazil. We detected M. leprae DNA in 32 (41 %) of the 78 individuals and M. lepromatosis DNA was not identified in any of the examined samples. The zoonotic component of leprosy may play a role in the transmission of the disease in endemic areas in which environmental conditions and contact with reservoirs must be investigated., 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. The author is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for [ActaTropica] and was not involved in the editorial review or the decision to publish this article. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Financial support was provided by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code: CAPES Epidemias 0670/2020, Number 88,881.505199/2020–01, Brasília, Brazil. Postdoctoral Scholarship by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) 88,887.671152/2022–00 (Lara, JM). Postdoctoral Scholarship by The São Paulo Research Foundation (FAPESP) 2018/12,565–6 (Drummond, MR); Ph.D. scholarship by The Brazilian National Council for Scientific and Technological Development (CNPq) 142,046/2019–1 (Silva, DP); Productivity Grant by The Brazilian National Council for Scientific and Technological Development (CNPq) 306,970/2018–0 (Velho, PENF) and 305,778/2021–8 (Donalisio, MR)., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.

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.)

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.)

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.

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.)

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.)

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 EC 50 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.

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.)

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., (Copyright © 2021 The Author(s). Published by Elsevier B.V. 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.)

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.

Acyclovir is the gold standard drug for herpes simplex virus type 2 (HSV-2) infection treatment. Vaginal topical therapy with acyclovir is hampered due to its poor bioavailability, low retention at the vaginal mucosa, thus requiring high doses and frequent administrations. Nanocarriers have been proposed to overcome the challenges associated with antiviral delivery. This work aims at developing a novel formulation consisting of sulfobutyl ether-β-cyclodextrin decorated nanodroplets for acyclovir topical delivery to improve its antiviral effectiveness. To obtain acyclovir-loaded nanodroplets, the drug was previously complexed with sulfobutyl ether-β-cyclodextrin, and then incorporated in the nanodroplet chitosan shell via electrostatic interaction. The acyclovir-cyclodextrin inclusion complex was characterized by phase solubility, DSC, FTIR studies. The nanodroplets showed an average diameter of about 400 nm and positive surface charge. Acyclovir was efficiently incorporated in the nanodroplets (about 97% of encapsulation efficiency) and slowly released over time. The acyclovir-loaded nanodroplets exhibited an enhanced antiviral activity compared to the free drug against HSV-2 in cell cultures, which might be ascribed to a higher intracellular accumulation of the drug in nanodroplet-treated cells than in free acyclovir-treated cells. Based on these results, this new nanoformulation paves the way for the development of a future nanomicrobicide for the HSV-2 infections., 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 © 2020 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.)

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.)

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- _ (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.)

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.)

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.

Thymus capitatus represents 1 of the 5 Tunisian species of the genus Thymus, which has long‐standing use for flavouring and preserving several food products. Its constituents have been reported to endow antimicrobial properties, but little is known about their antiviral activities. The aim of this study was to examine the antiviral activity of pure compounds from the most bioactive inhibitory T. capitatus extract in vitro against herpes simplex virus Type 2 (HSV‐2) infection and to identify their mechanism of action. Either the extracts or the essential oil exert inhibitory activity against HSV‐2 infection, with the ethanolic extract showing the lowest EC50 value (2.3 μg/ml). Three pure compounds were then isolated from the ethanolic extract and investigated for their antiviral activity. β‐sitosterol showed the most favourable selectivity index and both cinnamaldehyde and carvacrol exerted moderate antiviral effect. Investigation of the mechanism of action revealed that all three compounds directly inactivated the infectivity of the virus particles. These findings suggest the use of T. capitatus ethanolic extract as source of anti‐HSV‐2 pure compounds and warrant further studies to evaluate their therapeutic potential.