Emerging Strategies to Combat Methicillin-resistant Staphylococcus aureus (MRSA): Natural Agents with High Potential.

Infectious diseases have always been a concern for human health, responsible for numerous pandemics throughout history. Even with the advancement of medicine, new infectious diseases have been discovered over the years, requiring constant effort in medical research to avoid future problems. Like the emergence of new diseases, the increase in resistance of certain bacterial strains also becomes a concern, carried out through the misuse of antibiotics, generating the adaptation of certain microorganisms. Worldwide, the resistance developed by several bacterial strains is growing exponentially, creating awareness and developing novel strategies to control their evolution a mandatory research topic. Methicillin-resistant Staphylococcus aureus (MRSA) is an example of a bacterial strain that causes serious and mortal infections. The fact is that this bacterial strain started to develop resistance against commonly used antibiotics, first to penicillin and against methicillin. Thus, the treatment against infections caused by MRSA is limited and difficult due to its capacity to develop defense mechanisms against the antibiotic's action. Given the urgency to find new alternatives, the scientific community has been developing interesting research regarding the exploitation of natural resources to discover bioactive molecules that are able to inhibit/kill MRSA. In this sense, several natural matrices, namely plants, have shown great potential against MRSA, due to the presence of phenolic compounds, molecules with high antimicrobial capacity due to their chemical structure and arrangement.

Sympathetic Neural Overdrive, Aortic Stiffening, Endothelial Dysfunction, and Impaired Exercise Capacity in Severe COVID-19 Survivors: A Mid-Term Study of Cardiovascular Sequelae.

BACKGROUND: COVID-19 has become a dramatic health problem during this century. In addition to high mortality rate, COVID-19 survivors are at increased risk for cardiovascular diseases 1-year after infection. Explanations for these manifestations are still unclear but can involve a constellation of biological alterations. We hypothesized that COVID-19 survivors compared with controls exhibit sympathetic overdrive, vascular dysfunction, cardiac morpho-functional changes, impaired exercise capacity, and increased oxidative stress. METHODS: Nineteen severe COVID-19 survivors and 19 well-matched controls completed the study. Muscle sympathetic nerve activity (microneurography), brachial artery flow-mediated dilation and blood flow (Doppler-Ultrasound), carotid-femoral pulse wave velocity (Complior), cardiac morpho-functional parameters (echocardiography), peak oxygen uptake (cardiopulmonary exercise testing), and oxidative stress were measured ~3 months after hospital discharge. Complementary experiments were conducted on human umbilical vein endothelial cells cultured with plasma samples from subjects. RESULTS: Muscle sympathetic nerve activity and carotid-femoral pulse wave velocity were greater and brachial artery flow-mediated dilation, brachial artery blood flow, E/e' ratio, and peak oxygen uptake were lower in COVID-19 survivors than in controls. COVID-19 survivors had lower circulating antioxidant markers compared with controls, but there were no differences in plasma-treated human umbilical vein endothelial cells nitric oxide production and reactive oxygen species bioactivity. Diminished peak oxygen uptake was associated with sympathetic overdrive, vascular dysfunction, and reduced diastolic function in COVID-19 survivors. CONCLUSIONS: Our study revealed that COVID-19 survivors have sympathetic overactivation, vascular dysfunction, cardiac morpho-functional changes, and reduced exercise capacity. These findings indicate the need for further investigation to determine whether these manifestations are persistent longer-term and their impact on the cardiovascular health of COVID-19 survivors.

Chitosan Nano/Microformulations for Antimicrobial Protection of Leather with a Potential Impact in Tanning Industry.

Tanned leather can be attacked by microorganisms. To ensure resistance to bacteria on leather surfaces, protection solutions need to be developed, addressing both environmental issues and economic viability. In this work, chitosan nano/microparticles (CNP) and chitosan/silver nano/microstructures (CSNP), containing silver nanoparticles around 17 nm size, were incorporated into leather, obtained from the industrial process. Low loads of chitosan-based nano/microformulations, 0.1% mass ratio, resulted in total bacteria reduction (100%) after 2 h towards Gram-positive Staphylococcus aureus, both with CNP and CSNP coatings. Otherwise, comparable tests with the Gram-negative bacteria, Klebsiella pneumoniae, Escherichia coli, showed no significant improvement under the coating acidic conditions. The antimicrobial activity was evaluated by standard test methods: (1) inhibition halo and (2) dynamic contact conditions. The developed protection of leather either with CNP or CSNP is much higher than the one obtained with a simple chitosan solution.

West Nile virus transmission potential in Portugal.

It is unclear whether West Nile virus (WNV) circulates endemically in Portugal. Despite the country's adequate climate for transmission, Portugal has only reported four human WNV infections so far. We performed a review of WNV-related data (1966-2020), explored mosquito (2016-2019) and land type distributions (1992-2019), and used climate data (1981-2019) to estimate WNV transmission suitability in Portugal. Serological and molecular evidence of WNV circulation from animals and vectors was largely restricted to the south. Land type and climate-driven transmission suitability distributions, but not the distribution of WNV-capable vectors, were compatible with the North-South divide present in serological and molecular evidence of WNV circulation. Our study offers a comprehensive, data-informed perspective and review on the past epidemiology, surveillance and climate-driven transmission suitability of WNV in Portugal, highlighting the south as a subregion of importance. Given the recent WNV outbreaks across Europe, our results support a timely change towards local, active surveillance.

Synthesis of novel sugar derived aziridines, as starting materials giving access to sugar amino acid derivatives.

D-Erythrosyl aziridines were obtained from D-erythrosyl triazoles either by photolysis or through diazirine intermediates. These were found to undergo rich, high yielding chemistry by reaction with protic acids (HCl, BiI3/H2O and trifluoroacetic acid) leading to two types of furanoid sugar α-amino acids, and polyhydroxylprolines. Based on experimental evidence, reaction mechanisms have been proposed for the syntheses.

Effect of exercise training on cardiovascular autonomic and muscular function in subclinical Chagas cardiomyopathy: a randomized controlled trial.

PURPOSE: Patients with chronic chagasic cardiomyopathy with preserved ventricular function present with autonomic imbalance. This study evaluated the effects of exercise training (ET) in restoring peripheral and cardiac autonomic control and skeletal muscle phenotype in patients with subclinical chronic chagasic cardiomyopathy. METHODS: This controlled trial (NCT02295215) included 24 chronic chagasic cardiomyopathy patients who were randomized www.random.org/lists/ into two groups: those who underwent exercise training (n = 12) and those who continued their usual activities (n = 12). Eight patients completed the exercise training protocol, and 10 patients were clinically followed up for 4 months. Muscular sympathetic nerve activity was measured by microneurography and muscle blood flow (MBF) using venous occlusion plethysmography. The low-frequency component of heart rate variability in normalized units (LFnuHR) reflects sympathetic activity in the heart, and the low-frequency component of systolic blood pressure variability in normalized units reflects sympathetic activity in the vessels. The infusion of vasoactive drugs (phenylephrine and sodium nitroprusside) was used to evaluate cardiac baroreflex sensitivity, and a vastus lateralis muscle biopsy was performed to evaluate atrogin-1 and MuRF-1 gene expression. RESULTS: The baroreflex sensitivity for increases (p = 0.002) and decreases (p = 0.02) in systolic blood pressure increased in the ET group. Muscle blood flow also increased only in the ET group (p = 0.004). Only the ET group had reduced resting muscular sympathetic nerve activity levels (p = 0.008) and sympathetic activity in the heart (LFnu; p = 0.004) and vessels (p = 0.04) after 4 months. Regarding skeletal muscle, after 4 months, participants in the exercise training group presented with lower atrogin-1 gene expression than participants who continued their activities as usual (p = 0.001). The reduction in muscular sympathetic nerve activity was positively associated with reduced atrogin-1 (r = 0.86; p = 0.02) and MuRF-1 gene expression (r = 0.64; p = 0.06); it was negatively associated with improved baroreflex sensitivity both for increases (r = -0.72; p = 0.020) and decreases (r = -0.82; p = 0.001) in blood pressure. CONCLUSIONS: ET improved cardiac and peripheral autonomic function in patients with subclinical chagasic cardiomyopathy. ET reduced MSNA and sympathetic activity in the heart and vessels and increased cardiac parasympathetic tone and baroreflex sensitivity. Regarding peripheral muscle, after 4 months, patients who underwent exercise training had an increased cross-sectional area of type I fibers and oxidative metabolism of muscle fibers, and decreased atrogin-1 gene expression, compared to participants who continued their activities as usual. In addition, the reduction in MSNA was associated with improved cardiac baroreflex sensitivity, reduced sympathetic cardiovascular tone, and reduced atrogin-1 and MuRF-1 gene expression. TRIAL REGISTRATION: ID: NCT02295215. Registered in June 2013.

Adjuvant Treatment with 5-Fluorouracil and Oxaliplatin Does Not Influence Cardiac Function, Neurovascular Control, and Physical Capacity in Patients with Colon Cancer.

BACKGROUND: Adjuvant chemotherapy with 5-fluorouracil (5-FU) and oxaliplatin increases recurrence-free and overall survival in patients with colon adenocarcinoma. It is known that these drugs have been associated with cardio- and neurotoxicity. We investigated the effects of 5-FU ± oxaliplatin on cardiac function, vascular responses, neurovascular control, and physical capacity in patients with colon cancer. METHODS: Twenty-nine patients with prior colectomy for stage II-III adenocarcinoma and clinical indication for adjuvant chemotherapy were allocated to receive 5-FU (n = 12) or 5-FU + oxaliplatin (n = 17), according to the oncologist's decision. All the analyses were performed just before and after the end of chemotherapy. Cardiac function was assessed by echocardiography and speckle tracking, and cardiac autonomic control was assessed by heart rate variability (HRV). Vascular endothelial function was assessed by flow-mediated dilation (FMD). Muscle sympathetic nerve activity (MSNA) was directly recorded by microneurography technique, and muscle blood flow by venous occlusion plethysmography. Physical capacity was evaluated by cardiopulmonary exercise test. RESULTS: Chemotherapy (pooled data) did not significantly change left ventricular ejection fraction (58 ± 1 vs. 55 ± 2%, p = .14), longitudinal strain (-18 ± 1 vs. -18 ± 1%, p = .66), and HRV. Likewise, chemotherapy did not significantly change FMD, muscle blood flow, and MSNA (33 ± 2 vs. 32 ± 1 bursts/min, p = .31). Physical capacity was not significantly changed in both groups. Similar findings were observed when the patients were subdivided in 5-FU and 5-FU + oxaliplatin treatment groups. 5-FU and 5-FU + oxaliplatin did not significantly change cardiac function, HRV, vascular responses, MSNA, and physical capacity. CONCLUSION: This study provides evidence that adjuvant treatment with 5-FU ± oxaliplatin is well tolerated and does not promote changes compatible with long-term cardiotoxicity. IMPLICATIONS FOR PRACTICE: Adjuvant chemotherapy with 5-fluorouracil (5-FU) and oxaliplatin increases recurrence-free and overall survival in patients with colon adenocarcinoma; however, these drugs have been associated with cardio- and neurotoxicity. This study investigated the effects of these drugs on cardiac function, vascular responses, neurovascular control, and physical capacity in patients with colon cancer. It was found that 5-FU and oxaliplatin did not significantly change cardiac function, cardiac autonomic control, vascular endothelial function, muscle sympathetic nerve activity, and physical capacity. This study provides evidence that adjuvant treatment with 5-FU ± oxaliplatin is well tolerated and does not promote changes compatible with long-term cardiotoxicity.

Exercise training reverses cancer-induced oxidative stress and decrease in muscle COPS2/TRIP15/ALIEN.

OBJECTIVE: We tested the hypothesis that exercise training would attenuate metabolic impairment in a model of severe cancer cachexia. METHODS: We used multiple in vivo and in vitro methods to explore the mechanisms underlying the beneficial effects induced by exercise training in tumor-bearing rats. RESULTS: Exercise training improved running capacity, prolonged lifespan, reduced oxidative stress, and normalized muscle mass and contractile function in tumor-bearing rats. An unbiased proteomic screening revealed COP9 signalosome complex subunit 2 (COPS2) as one of the most downregulated proteins in skeletal muscle at the early stage of cancer cachexia. Exercise training normalized muscle COPS2 protein expression in tumor-bearing rats and mice. Lung cancer patients with low endurance capacity had low muscle COPS2 protein expression as compared to age-matched control subjects. To test whether decrease in COPS2 protein levels could aggravate or be an intrinsic compensatory mechanism to protect myotubes from cancer effects, we performed experiments in vitro using primary myotubes. COPS2 knockdown in human myotubes affected multiple cellular pathways, including regulation of actin cytoskeleton. Incubation of cancer-conditioned media in mouse myotubes decreased F-actin expression, which was partially restored by COPS2 knockdown. Direct repeat 4 (DR4) response elements have been shown to positively regulate gene expression. COPS2 overexpression decreased the DR4 activity in mouse myoblasts, and COPS2 knockdown inhibited the effects of cancer-conditioned media on DR4 activity. CONCLUSIONS: These studies demonstrated that exercise training may be an important adjuvant therapy to counteract cancer cachexia and uncovered novel mechanisms involving COPS2 to regulate myotube homeostasis in cancer cachexia.

Bacterial Resistance: Antibiotics of Last Generation used in Clinical Practice and the Arise of Natural Products as New Therapeutic Alternatives.

Bacterial resistance to therapeutical drugs has been a serious issue over the last decades. In fact, the quick development of resistance mechanisms by the microorganisms has been fatal for millions of people around the world, turning into a public health issue. The major cause of the resistance mechanisms is the overuse of antimicrobials. European countries try to implement mechanisms to overcome antimicrobial resistance in the community through the rational use of antimicrobials. The scientific community has been exhaustively dedicated to the discovering of new, safer and efficient drugs, being the exploitation of natural resources, mainly plants and fungi, considered as a hot topic in the field of antimicrobial agents. Innumerous reports have already shown the promising capacity of natural products or molecules extracted from these natural resources, to act as bacteriostatic and bactericidal agents. More importantly, these natural agents present significantly lower harmful effects. Bearing that in mind, this review aims at giving a contribution to the knowledge about the synthetic antibiotics of the last generation. Moreover, it is intended to provide information about the last advances regarding the discovery of new antimicrobial agents. Thus, a compilation of the chemical characteristics, efficiency, harmful outcomes and resistance mechanisms developed by the microorganisms can be consulted in the following sections together with a critical discussion, in line with the recent approaches. Furthermore, modern strategies for the prospection of novel anti-infective compounds for tackling resistant bacteria have been considered as also a current synopsis of plants and mushrooms with relevant antimicrobial potentials.

(3S,4R)-3,4-Dihydroxy-N-alkyl-l-homoprolines: synthesis and computational mechanistic studies.

This is the first synthetic report of (3S,4R)-dihydroxy-N-alkyl-l-homoprolines described so far. 2,4-O-Benzylidene-d-erythrose was obtained from d-glucose with an improved yield, and then transformed into the title (3S,4R)-dihydroxy-N-alkyl-l-homoprolines, in a two-step strategy, with excellent overall yields. Hydrogenolysis of the benzyl group led to the NH congener. The synthesis of final products from 1,4-lactone intermediates was studied by computational means either under acidic or basic conditions. The theoretical mechanism studies fully explain the experimental results: (a) an equilibrium between l-homoprolines and their bicyclic counterparts is established in acids; (b) the equilibrium suffers a complete displacement towards the l-homoproline side in a basic medium.

The Application and Interpretation of IgG Avidity and IgA ELISA Tests to Characterize Zika Virus Infections.

In the absence of viremia, the diagnostics of Zika virus (ZIKV) infections must rely on serological techniques. In order to improve the serological diagnosis of ZIKV, ZIKV-IgA and ZIKV-IgG avidity assays were evaluated. Forty patients returning from ZIKV endemic areas, with confirmed or suspected ZIKV infections were studied. Samples were classified as early acute, acute and late acute according to the number of days post illness onset. Low avidity IgG was only detected at acute and late acute stages and IgA mostly at the early acute and acute stages. The date of sampling provides useful information and can help to choose the best technique to use at a determined moment in time and to interpret low avidity IgG and IgA results, improving the serological diagnosis of ZIKV.

Generating Conformation-Specific Polyclonal and Monoclonal Anti-Protein Kinase C Antibodies and Anti-Active State Specific PKC Antibodies.

The protein kinase C (PKC) family of serine/ threonine kinases has been shown to play active roles as either suppressors or promoters of carcinogenesis in different types of tumors. Using antibodies that preferentially recognize the active conformation of classical PKCs (cPKCs), we have previously shown that in breast cancer samples the expression levels of cPKCs were similar in estrogen receptor-positive (ER+ ) as compared to triple-negative tumors; however, the levels of active cPKCs were different. Determining the activation status of PKCs and other kinases in tumors may thus aid therapeutic decisions. Further, in basic science these tools may be used to understand the spatial and temporal dynamics of PKC signaling under different stimuli and for co-immunoprecipitation studies to detect binding partners and substrates of active cPKCs. In this article, we describe how monoclonal and polyclonal anti-active state PKC antibodies can be obtained using rational approaches to select bona fide epitopes through inspection of the crystal structure of classical PKCs coupled to molecular modeling studies. We believe that this methodology can be used for other kinases and multi-domain enzymes that undergo changes in their conformation upon activation. © 2018 by John Wiley & Sons, Inc.

Total Stereoselective Michael Addition of N- and S- Nucleophiles to a d-Erythrosyl 1,5-Lactone Derivative. Experimental and Theoretical Studies Devoted to the Synthesis of 2,6-Dideoxy-4-functionalized-d- ribono-hexono-1,4-lactone.

The synthesis of a 1,5-lactone 2,4- O-alkylidene-d-erythrose derivative was found to be a highly stereoselective template in Michael addition trough the reaction of a d-erythrosyl 1,5-lactone derivative with nitrogen and sulfur nucleophiles. The sulfur adducts formed are 1 (d-erythrose derivative):1 (nucleophile), and the nitrogen adducts are 1:2. Both were then treated under HCl to give 2,6-dideoxy-4-functionalized-d- ribono-hexono-1,4-lactone by a reaction cascade in high overall yield. Reaction's scale up even improves the yield. The theoretical and computational results clearly explain the origin of the stereoselectivity, and the energetic course of reactions starting with nitrogen and sulfide nucleophiles. Considering that the 1,4-lactones obtained in this work offer a new molecular scaffold for organic synthesis, these new results provide a solid theoretical platform that can be used to speed up synthesis of other derivatives in a stereo- and regioselective way.

Muscle Metaboreflex Control of Sympathetic Activity in Obstructive Sleep Apnea.

PURPOSE: Previous studies report abnormal muscle metaboreflex control of muscle sympathetic nerve activity (MSNA) in obesity, hypertension, and heart failure. We hypothesized that obstructive sleep apnea (OSA) is associated with augmented metaboreflex control of MSNA. METHODS: Thirty-one sedentary individuals with no comorbidities (age = 52 ± 1 yr, body mass index = 28 ± 1 kg·m) without (control, n = 14) and with OSA (n = 17) defined by polysomnography, underwent echocardiography. HR, blood pressure (BP), MSNA (microneurography), and forearm blood flow measured by venous occlusion plethysmography were continuously measured 4 min at baseline, during 3 min of 30% handgrip static exercise, and during 2 min of post-handgrip muscle ischemia (PHMI). RESULTS: Control and OSA groups were similar in age, body mass index, and ejection fraction. Baseline HR, BP, and forearm blood flow increased similarly during handgrip exercise. Blood pressure remained significantly elevated in relation to baseline during PHMI, but HR and forearm blood flow returned toward baseline during PHMI in both groups. Baseline MSNA was significantly higher in the OSA group than in controls (P < 0.05). During peak 30% static handgrip exercise, MSNA increased significantly in both control and OSA groups, but MSNA responses were higher in patients with OSA. During PHMI, MSNA in control subjects remained significantly elevated compared with that at baseline. In contrast, in patients with OSA, MSNA decreased to baseline values. A significant correlation was found between changes in MSNA due to PHMI and apnea-hypopnea index (r = -0.61, P < 0.001), and with minimum O2 saturation (r = 0.70, P < 0.001). CONCLUSIONS: These findings suggest an association between OSA and decreased metaboreflex control of MSNA. Muscle vasodilation during handgrip static exercise is preserved in patients with OSA.

Total Facial Discrimination of 1,3-Dipolar Cycloadditions in a d-Erythrose 1,3-Dioxane Template: Computational Studies of a Concerted Mechanism.

A new d-erythrose 1,3-dioxane derivative was synthesized from d-glucose and found to be a highly stereoselective template as a dipolarophile. Different 1,3-dipoles of allenyl-type were employed, giving different regioselectivities, depending on its nature; the regioselectivity is complete with alkyl azides and phenyldiazomethane, but is inexistence with nitrile oxides. Computational studies were performed to understand the mechanisms of cycloadditions. All the studied cycloadditions were found to be concerted involving small free activation energies and are all exoenergonic. The stereoselectivity is due to a combined result of the steric effect H-8a and the hyperconjugative effect of the *C-O to the incoming 1,3-dipole. The regioselectivity observed in alkyl azides and phenyldiazomethane is mostly dependent on the distortion effect during the cycloaddition process. This distortion effect is however higher in the alkyl azide compounds than in phenyldiazomethane.

Total facial selectivity of a D-erythrosyl aromatic imine in [4π + 2π] cycloadditions; synthesis of 2-alkylpolyol 1,2,3,4-tetrahydroquinolines.

Different electron-rich dienophiles were combined with the imine obtained from 2,4-O-benzylidene-d-erythrose and p-anisidine furnishing enantiomerically pure tetrahydroquinolines, by inverse electron-demand [4π + 2π] cycloaddition. The imine was also reacted with 2-substituted electron-rich 1,3-butadienes giving the diastereomeric pure product, resulting from the normal electron demand cycloaddition. The facial selectivity of both processes is proposed on the basis of a 1,4-relationship between the hydroxyl group and the nitrogen atom in the chiral N-(p-methoxyphenyl)imine derivative.

Vesicles from different Trypanosoma cruzi strains trigger differential innate and chronic immune responses.

Trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas Disease, shed extracellular vesicles (EVs) enriched with glycoproteins of the gp85/trans-sialidase (TS) superfamily and other α-galactosyl (α-Gal)-containing glycoconjugates, such as mucins. Here, purified vesicles from T. cruzi strains (Y, Colombiana, CL-14 and YuYu) were quantified according to size, intensity and concentration. Qualitative analysis revealed differences in their protein and α-galactosyl contents. Later, those polymorphisms were evaluated in the modulation of immune responses (innate and in the chronic phase) in C57BL/6 mice. EVs isolated from YuYu and CL-14 strains induced in macrophages higher levels of proinflammatory cytokines (TNF-α and IL-6) and nitric oxide via TLR2. In general, no differences were observed in MAPKs activation (p38, JNK and ERK 1/2) after EVs stimulation. In splenic cells derived from chronically infected mice, a different modulation pattern was observed, where Colombiana (followed by Y strain) EVs were more proinflammatory. This modulation was independent of the T. cruzi strain used in the mice infection. To test the functional importance of this modulation, the expression of intracellular cytokines after in vitro exposure was evaluated using EVs from YuYu and Colombiana strains. Both EVs induced cytokine production with the appearance of IL-10 in the chronically infected mice. A high frequency of IL-10 in CD4+ and CD8+ T lymphocytes was observed. A mixed profile of cytokine induction was observed in B cells with the production of TNF-α and IL-10. Finally, dendritic cells produced TNF-α after stimulation with EVs. Polymorphisms in the vesicles surface may be determinant in the immunopathologic events not only in the early steps of infection but also in the chronic phase.

Co-circulation of a novel phlebovirus and Massilia virus in sandflies, Portugal.

BACKGROUND: In Portugal, entomological surveys to detect phleboviruses in their natural vectors have not been performed so far. Thus, the aims of the present study were to detect, isolate and characterize phleboviruses in sandfly populations of Portugal. FINDINGS: From May to October 2007-2008, 896 female sandflies were trapped in Arrábida region, located on the southwest coast of Portugal. Phlebovirus RNA was detected by using a pan-phlebovirus RT-PCR in 4 out of 34 Phlebotomus perniciosus pools. Direct sequencing of the amplicons showed that 2 samples exhibited 72 % nucleotide identity with Arbia virus, and two showed 96 % nucleotide identity with Massilia virus. The Arbia-like virus (named Alcube virus) was isolated in cell culture and complete genomic sequences of one Alcube and two Massila viruses were determined using next-generation sequencing technology. Phylogenetic analysis demonstrated that Alcube virus clustered with members of the Salehabad virus species complex. Within this clade, Alcube virus forms a monophyletic lineage with the Arbia, Salehabad and Adana viruses sharing a common ancestor. Arbia virus has been identified as the most closely related virus with 20-28 % nucleotide and 10-27 % amino acid divergences depending on the analysed segment. CONCLUSIONS: We have provided genetic evidence for the circulation of a novel phlebovirus species named Alcube virus in Ph. perniciosus and co-circulation of Massilia virus, in Arrábida region, southwest of Portugal. Further epidemiological investigations and surveillance for sandfly-borne phleboviruses in Portugal are needed to elucidate their medical importance.