Human Colostrum and Derived Extracellular Vesicles Prevent Infection by Human Rotavirus and Respiratory Syncytial Virus in Vitro.

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.

Punica granatum Leaf Ethanolic Extract and Ellagic Acid as Inhibitors of Zika Virus Infection.

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.

Antiviral Activity of a Arisaema Tortuosum Leaf Extract and Some of its Constituents against Herpes Simplex Virus Type 2.

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.

Antiviral oxysterols are present in human milk at diverse stages of lactation.

Oxysterols are cholesterol oxidation derivatives. Those containing an additional hydroxyl group on the side chain of the cholesterol molecule result from a physiological enzymatic synthesis and include the majority of oxysterols present in the circulation. Among these, 25-hydroxycholesterol (25OHC) and 27-hydroxycholesterol (27OHC) are characterized by a broad antiviral activity and are now considered involved in the innate immune response against viruses. Despite the emerging role of these sterols in the innate antiviral defences, no data are available on their presence in human breast milk (BM) to date. In this study, we investigated the content of oxysterols of enzymatic synthesis in BM of twelve donor mothers at different stages of lactation (i.e. in colostrum, transitional milk, and mature milk) by gas chromatography-mass spectrometry analysis. The side-chain oxysterols 25OHC, 27OHC, and 24S-hydroxycholesterol (24SOHC) were actually present in BM in all stages of lactation, but the concentration of 27OHC showed a remarkable peak in colostrum. Antiviral assays revealed that all the colostrum samples contained 27OHC concentrations that were active in vitro against two relevant pediatric viral pathogens: the human rotavirus and the human rhinovirus. Overall, this study discloses new antiviral components of BM and suggests a passive transfer of these protective factors to the infant via breastfeeding, especially in the first few days of lactation.

Colostrum from cows immunized with a veterinary vaccine against bovine rotavirus displays enhanced in vitro anti-human rotavirus activity.

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.

Anti-Cytomegalovirus Activity in Human Milk and Colostrum From Mothers of Preterm Infants.

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.

Inhibition of HSV-2 infection by pure compounds from Thymus capitatus extract in vitro.

Thymus capitatus represents 1 of the 5 Tunisian species of the genus Thymus, which has long­standing 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 (HSV­2) infection and to identify their mechanism of action. Either the extracts or the essential oil exert inhibitory activity against HSV­2 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. ß­sitosterol 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 anti­HSV­2 pure compounds and warrant further studies to evaluate their therapeutic potential.

The traditional use of Vachellia nilotica for sexually transmitted diseases is substantiated by the antiviral activity of its bark extract against sexually transmitted viruses.

ETHNOPHARMACOLOGICAL RELEVANCE: Vachellia (Acacia) nilotica and other plants of this genus have been used in traditional medicine of Asian and African countries to treat many disorders, including sexually transmitted diseases, but few studies were performed to validate their anti-microbial and anti-viral activity against sexually transmitted infections. AIM OF THE STUDY: The present study was undertaken to explore whether the ethnomedical use of V.nilotica to treat genital lesions is substantiated by its antiviral activity against the human immunodeficiency virus (HIV), the herpes simplex virus (HSV) and the human papillomavirus (HPV). MATERIALS AND METHODS: The antiviral activity of V.nilotica was tested in vitro by virus-specific inhibition assays using HSV-2 strains, sensible or resistant to acyclovir, HIV-1IIIb strain and HPV-16 pseudovirion (PsV). The potential mode of action of extract against HSV-2 and HPV-16 was further investigated by virus inactivation and time-of-addition assays on cell cultures. RESULTS: V.nilotica chloroform, methanolic and water bark extracts exerted antiviral activity against HSV-2 and HPV-16 PsV infections; among these, methanolic extract showed the best EC50s with values of 4.71 and 1.80µg/ml against HSV-2 and HPV-16, respectively, and it was also active against an acyclovir-resistant HSV-2 strain with an EC50 of 6.71µg/ml. By contrast, no suppression of HIV infection was observed. Investigation of the mechanism of action revealed that the methanolic extract directly inactivated the infectivity of the HPV-16 particles, whereas a partial virus inactivation and interference with virus attachment (EC50 of 2.74µg/ml) were both found to contribute to the anti-HSV-2 activity. CONCLUSIONS: These results support the traditional use of V.nilotica applied externally for the treatment of genital lesions. Further work remains to be done in order to identify the bioactive components.

In vitro anti-herpes simplex virus-2 activity of Salvia desoleana Atzei & V. Picci essential oil.

Salvia desoleana Atzei & V. Picci is an indigenous species in Sardinia island used in folk medicine to treat menstrual, digestive and central nervous system diseases. Nowadays, it is widely cultivated for the pleasant smell of its essential oil (EO), whose antimicrobial and antifungal activities have already been screened. This study evaluated the in vitro anti-Herpes Simplex Virus-2 (HSV-2) activity of S. desoleana EO, fractions and main components: linalyl acetate, alpha terpinyl acetate, and germacrene D. Phytochemical composition of S. desoleana EO was studied by GC-FID/MS analysis and the active fraction(s) and/or compounds in S. desoleana EO were identified with a bioassay-guided fractionation procedure through in vitro assays on cell viability and HSV-2 and RSV inhibition. S. desoleana EO inhibits both acyclovir sensitive and acyclovir resistant HSV-2 strains with EC50 values of 23.72 µg/ml for the former and 28.57 µg/ml for the latter. Moreover, a significant suppression of HSV-2 replication was observed with an EC50 value of 33.01 µg/ml (95% CI: 26.26 to 41.49) when the EO was added post-infection. Among the fractions resulting from flash column chromatography on silica gel, the one containing 54% of germacrene D showed a similar spectrum of activity of S. desoleana EO with a stronger suppression in post-infection stage. These results indicated that S. desoleana EO can be of interest to develop new and alternative anti-HSV-2 products active also against acyclovir-resistant HSV-2 strains.

In vitro screening for antiviral activity of Turkish plants revealing methanolic extract of Rindera lanata var. lanata active against human rotavirus.

BACKGROUND: Human rotavirus (HRoV) is the leading cause of severe gastroenteritis in infants and children under the age of five years. No specific antiviral drug is available for HRoV infections and the treatment of viral diarrhea is mainly based on rehydration and zinc treatment. In this study, we explored medicinal plants endemic to Turkey flora as a source of anti-HRoV compunds. METHODS: We performed an antiviral screening on Ballota macrodonta, Salvia cryptantha and Rindera lanata extracts by focus reduction assay. The extract with the highest selectivity index (SI) was selected; its antiviral activity was further confirmed against other HRoV strains and by virus yield reduction assay. The step of viral replicative cycle putatively inhibited was investigated by in vitro assays. RESULTS: The methanolic extract of R. lanata (Boraginaceae) showed the most favourable selectivity index. This extract exhibited a dose-dependent inhibitory activity against three different HRoV strains (EC50 values ranging from 5.8 µg/ml to 25.5 µg/ml), but was inactive or barely active against other RNA viruses, namely human rhinovirus and respiratory syncytial virus. The R. lanata extract targets the early steps of HRoV infection, likely by hampering virus penetration into the cells. CONCLUSION: These results make the R. lanata methanolic extract a promising starting material for a bioguided-fractionation aimed at identifying anti-HRoV compounds. Further work is required to isolate the active principle and assess its clinical potential.

Ficus religiosa L. bark extracts inhibit infection by herpes simplex virus type 2 in vitro.

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

Ficus religiosa L. bark extracts inhibit human rhinovirus and respiratory syncytial virus infection in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Ficus religiosa L. is one of the most relevant members of the family of Moraceae. It is the most sacred tree of South Asia, and it is used in traditional Ayurvedic and Unani medicine to cure respiratory disorders like cough, wheezing and asthma. Some studies were performed to investigate the anti-asthmatic potential of F. religiosa bark, leaves and fruit extracts but none of them tested their antiviral activity against viruses responsible for the exacerbation of wheezing and asthma. AIM OF THE STUDY: The present study was undertaken to investigate the antiviral activity of F. religiosa L. extracts against respiratory viruses such as human respiratory syncytial virus (RSV) and human rhinovirus (HRV). MATERIALS AND METHODS: The antiviral activity of F. religiosa L. was tested in vitro by plaque reduction and virus yield assays and the major mechanism of action was investigated by virus inactivation and time-of-addition assays. RESULTS: F. religiosa L. methanol bark extract was the most active against HRV with an EC50 of 5.52 µg/mL. This extract likely inhibited late steps of replicative cycle. Water bark extract was the most active against RSV with an EC50 between 2.23 and 4.37 µg/mL. Partial virus inactivation and interference with virus attachment were both found to contribute to the anti-RSV activity. Replication of both viruses was inhibited in viral yield reduction assays. CONCLUSIONS: The results of the present study demonstrate that F. religiosa L. is endowed with antiviral activity against RSV and HRV in vitro. Further work remains to be done to identify the active components and to assess the therapeutic potential in vivo.

In vitro evaluation of the antiviral properties of Shilajit and investigation of its mechanisms of action.

ETHNOPHARMACOLOGICAL RELEVANCE: Shilajit, a herbomineral substance exuded from rocks in steep mountainous regions, has been used for thousands of years by the Indian Ayurvedic and Siddha systems of traditional medicine to relieve ailments and enhance quality of life. Although a large number of therapeutic properties have been ascribed to Shilajit, its therapeutic potential is still largely unexplored by modern research and many of its claimed bioactivities lack scientific validation. The present study was undertaken to investigate the antiviral activity of Shilajit against a panel of viruses including herpes simplex type 1 and 2 (HSV-1, HSV-2), human cytomegalovirus (HCMV), human respiratory syncytial virus (RSV), human rotavirus (HRV), and vesicular stomatitis virus (VSV). MATERIALS AND METHODS: The antiviral activity of Shilajit was assayed in vitro by plaque reduction and virus yield assays and the major mechanism of action was investigated by virucidal and time-of-addition assays. RESULTS: Shilajit exhibited a dose-dependent inhibitory activity against HSV1, HSV2, HCMV, and RSV infectivity in vitro (EC50 values: 31.08µg/ml, 12.85µg/ml, 34.54µg/ml, and 30.35µg/ml, respectively), but was inactive against HRV and VSV. Humic acid, a constituent of Shilajit, displayed the same spectrum of activity. Partial virus inactivation and interference with virus attachment were both found to contribute to the antiviral activity of Shilajit. CONCLUSIONS: The results of the present study demonstrate that Shilajit is endowed with broad, yet specific, antiviral activity in vitro and constitutes a natural source of antiviral substances. Further work remains to be done to assess its efficacy in vivo.