Typing of Leishmania isolates from vectors and leporids of the Madrid (Spain) outbreak.

In 2009, a large outbreak of leishmaniasis, associated with environmental changes, was declared near Madrid (Spain), in which Phlebotomus perniciosus was the vector, whereas the main reservoirs were hares and rabbits. Analysis of isolates from humans, vectors and leporids from the focus identified the Leishmania infantum ITS-Lombardi genotype. However, multilocus enzyme electrophoresis (MLEE), the reference technique for Leishmania typing, and sequencing of the hsp70 gene, a commonly used marker, were not performed. In the present study, 19 isolates from P. perniciosus (n = 11), hares (n = 5) and rabbits (n = 3) from the outbreak area, all characterized as ITS-Lombardi in previous studies, were analysed by MLEE and hsp70 sequencing. The hsp70 results confirmed that all the analysed strains are L. infantum. However, by MLEE, 4 different zymodemes of L. infantum were identified based on variable mobilities of the NP1 enzyme: MON-34 (NP1100, n = 11), MON-80 (NP1130, n = 6), MON-24 (NP1140, n = 1) and MON-331 (NP1150, n = 1). The relative frequency of these zymodemes does not correspond to their usual occurrence in Spain. Moreover, MON-34 and MON-80 were found in P. perniciosus, hares and rabbits for the first time. These findings continue to provide insights into the outbreak and call for further studies with a higher number of strains.

Novel salivary antihemostatic activities of long-form D7 proteins from the malaria vector Anopheles gambiae facilitate hematophagy.

To successfully feed on blood, hematophagous arthropods must combat the host's natural hemostatic and inflammatory responses. Salivary proteins of blood-feeding insects such as mosquitoes contain compounds that inhibit these common host defenses against blood loss, including vasoconstriction, platelet aggregation, blood clotting, pain, and itching. The D7 proteins are some of the most abundantly expressed proteins in female mosquito salivary glands and have been implicated in inhibiting host hemostatic and inflammatory responses. Anopheles gambiae, the primary vector of malaria, expresses three D7 long-form and five D7 short-form proteins. Previous studies have characterized the AngaD7 short-forms, but the D7 long-form proteins have not yet been characterized in detail. Here, we characterized the A. gambiae D7 long-forms by first determining their binding kinetics to hemostatic agonists such as leukotrienes and serotonin, which are potent activators of vasoconstriction, edema formation, and postcapillary venule leakage, followed by ex vivo functional assays. We found that AngaD7L1 binds leukotriene C4 and thromboxane A2 analog U-46619; AngaD7L2 weakly binds leukotrienes B4 and D4; and AngaD7L3 binds serotonin. Subsequent functional assays confirmed AngaD7L1 inhibits U-46619-induced platelet aggregation and vasoconstriction, and AngaD7L3 inhibits serotonin-induced platelet aggregation and vasoconstriction. It is therefore possible that AngaD7L proteins counteract host hemostasis by scavenging these mediators. Finally, we demonstrate that AngaD7L2 had a dose-dependent anticoagulant effect via the intrinsic coagulation pathway by interacting with factors XII, XIIa, and XI. The uncovering of these interactions in the present study will be essential for comprehensive understanding of the vector-host biochemical interface.

A deeper insight into the sialome of male and female Culex quinquefasciatus mosquitoes.

INTRODUCTION: During evolution, blood-feeding arthropods developed a complex salivary mixture that can interfere with host haemostatic and immune response, favoring blood acquisition and pathogen transmission. Therefore, a survey of the salivary gland contents can lead to the identification of molecules with potent pharmacological activity in addition to increase our understanding of the molecular mechanisms underlying the hematophagic behaviour of arthropods. The southern house mosquito, Culex quinquefasciatus, is a vector of several pathogenic agents, including viruses and filarial parasites that can affect humans and wild animals. RESULTS: Previously, a Sanger-based transcriptome of the salivary glands (sialome) of adult C. quinquefasciatus females was published based on the sequencing of 503 clones organized into 281 clusters. Here, we revisited the southern mosquito sialome using an Illumina-based RNA-sequencing approach of both male and female salivary glands. Our analysis resulted in the identification of 7,539 coding DNA sequences (CDS) that were functionally annotated into 25 classes, in addition to 159 long non-coding RNA (LncRNA). Additionally, comparison of male and female libraries allowed the identification of female-enriched transcripts that are potentially related to blood acquisition and/or pathogen transmission. CONCLUSION: Together, these findings represent an extended reference for the identification and characterization of the proteins containing relevant pharmacological activity in the salivary glands of C. quinquefasciatus mosquitoes.

A mosquito juvenile hormone binding protein (mJHBP) regulates the activation of innate immune defenses and hemocyte development.

Insects rely on the innate immune system for defense against pathogens, some aspects of which are under hormonal control. Here we provide direct experimental evidence showing that the juvenile hormone-binding protein (mJHBP) of Aedes aegypti is required for the regulation of innate immune responses and the development of mosquito blood cells (hemocytes). Using an mJHBP-deficient mosquito line generated by means of CRISPR-Cas9 gene editing technology we uncovered a mutant phenotype characterized by immunosuppression at the humoral and cellular levels, which profoundly affected susceptibility to bacterial infection. Bacteria-challenged mosquitoes exhibited significantly higher levels of septicemia and mortality relative to the wild type (WT) strain, delayed expression of antimicrobial peptides (AMPs), severe developmental dysregulation of embryonic and larval hemocytes (reduction in the total number of hemocytes) and increased differentiation of the granulocyte lineage. Interestingly, injection of recombinant wild type mJHBP protein into adult females three-days before infection was sufficient to restore normal immune function. Similarly, injection of mJHBP into fourth-instar larvae fully restored normal larval/pupal hemocyte populations in emerging adults. More importantly, the recovery of normal immuno-activation and hemocyte development requires the capability of mJHBP to bind JH III. These results strongly suggest that JH III functions in mosquito immunity and hemocyte development in a manner that is perhaps independent of canonical JH signaling, given the lack of developmental and reproductive abnormalities. Because of the prominent role of hemocytes as regulators of mosquito immunity, this novel discovery may have broader implications for the understanding of vector endocrinology, hemocyte development, vector competence and disease transmission.

Regio- and Stereoselectivity of the Norrish-Yang Photocyclization of Dialkyl 1,2-Diketones: Solution versus Solid State Photochemistry of Two Polymorphs.

As shown by X-ray crystallography, crystals of 3ß-acetoxy-16,17-seco-17,20-dioxopregn-5-ene-16-nitrile are dimorphic. The regioselectivity of the Norrish-Yang type II photocyclization under visible light of this steroidal 1,2-diketone, which bears primary, secondary, and tertiary nonequivalent abstractable γ-hydrogens, dramatically increases in the crystalline state of both polymorphs. X-ray crystallography and molecular mechanics calculations reveal crystal structure-solid state photochemistry relationships.

Aedes aegypti SGS1 is critical for Plasmodium gallinaceum infection of both the mosquito midgut and salivary glands.

BACKGROUND: The invasion of the mosquito salivary glands by Plasmodium sporozoites is a critical step that defines the success of malaria transmission and a detailed understanding of the molecules responsible for salivary gland invasion could be leveraged towards control of vector-borne pathogens. Antibodies directed against the mosquito salivary gland protein SGS1 have been shown to reduce Plasmodium gallinaceum sporozoite invasion of Aedes aegypti salivary glands, but the specific role of this protein in sporozoite invasion and in other stages of the Plasmodium life cycle remains unknown. METHODS: RNA interference and CRISPR/Cas9 were used to evaluate the role of A. aegypti SGS1 in the P. gallinaceum life cycle. RESULTS: Knockdown and knockout of SGS1 disrupted sporozoite invasion of the salivary gland. Interestingly, mosquitoes lacking SGS1 also displayed fewer oocysts. Proteomic analyses confirmed the abolishment of SGS1 in the salivary gland of SGS1 knockout mosquitoes and revealed that the C-terminus of the protein is absent in the salivary gland of control mosquitoes. In silico analyses indicated that SGS1 contains two potential internal cleavage sites and thus might generate three proteins. CONCLUSION: SGS1 facilitates, but is not essential for, invasion of A. aegypti salivary glands by P. gallinaceum and has a dual role as a facilitator of parasite development in the mosquito midgut. SGS1 could, therefore, be part of a strategy to decrease malaria transmission by the mosquito vector, for example in a transgenic mosquito that blocks its interaction with the parasite.

1,5-Hydrogen Atom Transfer/Surzur-Tanner Rearrangement: A Radical Cascade Approach for the Synthesis of 1,6-Dioxaspiro[4.5]decane and 6,8-Dioxabicyclo[3.2.1]octane Scaffolds in Carbohydrate Systems.

The 1,5-HAT-1,2-(ester)alkyl radical migration (Surzur-Tanner rearrangement) radical/polar sequence triggered by alkoxyl radicals has been studied on a series of C-glycosyl substrates with 3-C-(α,ß-d,l-glycopyranosyl)1-propanol and C-(α-d,l-glycopyranosyl)methanol structures prepared from chiral pool d- and l-sugar. The use of acetoxy and diphenoxyphosphatoxy as leaving groups provides an efficient construction of 10-deoxy-1,6-dioxaspiro[4.5]decane and 4-deoxy-6,8-dioxabicyclo[3.2.1]octane frameworks. The alkoxyl radicals were generated by the reaction of the corresponding N-alkoxyphthalimides with group 14 hydrides [n-Bu3SnH(D) and (TMS)3SiH], and in comparative terms, the reaction was also initiated by visible light photocatalysis using the Hantzsch ester/fac-Ir(ppy)3 procedure. Special attention was devoted to the influence of the relative stereochemistry of the centers involved in the radical sequence on the reaction outcome. The addition of BF3•Et2O as a catalyst to the radical sequence resulted in a significant increase in the yields of the desired bicyclic ketals.

Aedes aegypti Piwi4 Structural Features Are Necessary for RNA Binding and Nuclear Localization.

The PIWI-interacting RNA (piRNA) pathway provides an RNA interference (RNAi) mechanism known from Drosophila studies to maintain the integrity of the germline genome by silencing transposable elements (TE). Aedes aegypti mosquitoes, which are the key vectors of several arthropod-borne viruses, exhibit an expanded repertoire of Piwi proteins involved in the piRNA pathway, suggesting functional divergence. Here, we investigate RNA-binding dynamics and subcellular localization of A. aegypti Piwi4 (AePiwi4), a Piwi protein involved in antiviral immunity and embryonic development, to better understand its function. We found that AePiwi4 PAZ (Piwi/Argonaute/Zwille), the domain that binds the 3' ends of piRNAs, bound to mature (3' 2' O-methylated) and unmethylated RNAs with similar micromolar affinities (KD = 1.7 ± 0.8 µM and KD of 5.0 ± 2.2 µM, respectively; p = 0.05) in a sequence independent manner. Through site-directed mutagenesis studies, we identified highly conserved residues involved in RNA binding and found that subtle changes in the amino acids flanking the binding pocket across PAZ proteins have significant impacts on binding behaviors, likely by impacting the protein secondary structure. We also analyzed AePiwi4 subcellular localization in mosquito tissues. We found that the protein is both cytoplasmic and nuclear, and we identified an AePiwi4 nuclear localization signal (NLS) in the N-terminal region of the protein. Taken together, these studies provide insights on the dynamic role of AePiwi4 in RNAi and pave the way for future studies aimed at understanding Piwi interactions with diverse RNA populations.

Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection.

Salivary components from disease vectors help arthropods to acquire blood and have been shown to enhance pathogen transmission in different model systems. Here we show that two salivary enzymes from Lutzomyia longipalpis have a synergist effect that facilitates a more efficient blood meal intake and diffusion of other sialome components. We have previously shown that Lundep, a highly active endonuclease, enhances parasite infection and prevent blood clotting by inhibiting the intrinsic pathway of coagulation. To investigate the physiological role of a salivary hyaluronidase in blood feeding we cloned and expressed a recombinant hyaluronidase from Lu. longipalpis. Recombinant hyaluronidase (LuloHya) was expressed in mammalian cells and biochemically characterized in vitro. Our study showed that expression of neutrophil CXC chemokines and colony stimulating factors were upregulated in HMVEC cells after incubation with LuloHya and Lundep. These results were confirmed by the acute hemorrhage, edema and inflammation in a dermal necrosis (dermonecrotic) assay involving a massive infiltration of leukocytes, especially neutrophils, in mice co-injected with hemorrhagic factor and these two salivary proteins. Moreover, flow cytometry results showed that LuloHya and Lundep promote neutrophil recruitment to the bite site that may serve as a vehicle for establishment of Leishmania infection. A vaccination experiment demonstrated that LuloHya and Lundep confer protective immunity against cutaneous leishmaniasis using the Lu. longipalpis-Leishmania major combination as a model. Animals (C57BL/6) immunized with LuloHya or Lundep showed minimal skin damage while lesions in control animals remained ulcerated. This protective immunity was abrogated when B-cell-deficient mice were used indicating that antibodies against both proteins play a significant role for disease protection. Rabbit-raised anti-LuloHya antibodies completely abrogated hyaluronidase activity in vitro. Moreover, in vivo experiments demonstrated that blocking LuloHya with specific antibodies interferes with sand fly blood feeding. This work highlights the relevance of vector salivary components in blood feeding and parasite transmission and further suggests the inclusion of these salivary proteins as components for an anti-Leishmania vaccine.

Synthetic Approaches to Phosphasugars (2-oxo-1,2-oxaphosphacyclanes) Using the Anomeric Alkoxyl Radical ß-Fragmentation Reaction as the Key Step.

The anomeric alkoxyl radical ß-fragmentation (ARF) of carbohydrates possessing an electron-withdrawing group (EWG) at C2, promoted by PhI(OAc)2/I2, gives rise to an acyclic iodide through which a pentavalent atom of phosphorus can be introduced via the Arbuzov reaction. After selective hydrolysis and subsequent cyclization, the phosphonate or phosphinate intermediates can be converted into 2-deoxy-1-phosphahexopyranose and 2-deoxy-1-phosphapentopyranose sugars. The ARF of carbohydrates with an electron-donor group (EDG) at C2 proceeds by a radical-polar crossover mechanism, and the cyclization occurs by nucleophilic attack of a conveniently positioned phosphonate or phosphinate group to the transient oxocarbenium ion. This alternative methodology leads to 5-phosphasugars with a 4-deoxy-5-phosphapentopyranose framework. The structure and conformation of the 2-oxo-1,2-oxaphosphinane and 2-oxo-1,2-oxaphospholane ring systems in different carbohydrate models have been studied by NMR and X-ray crystallography.

Pomegranate (Punica granatum) Seed Oil for Treating Menopausal Symptoms: An Individually Controlled Cohort Study.

Context • In the folk medicine of Mediterranean countries and in ancient Ayurveda, Punica granatum seeds (ie, pomegranate seeds) have been used for treatment of various disorders, including those that nowadays are classified as menopausal symptoms (MSs). Pomegranate seed oil (PSO) from those seeds mainly contains unsaturated fatty acids such as γ-linoleic acid and linolenic acid, but it also includes phytoestrogens. It is, therefore, regarded as a promising option for treating MSs today. Objectives • The study intended to investigate the safety and effectiveness of PSO as a defined P granatum seed oil for patients with MSs. Design • The research team designed an individually controlled, investigator-initiated cohort study. Setting • The treatments were performed at 2 institutions: (1) the Center for Complementary Medicine at the University Medical Center Freiburg (Freiburg, Germany); and (2) in the medical practice of H. Fischer (Freiburg, Germany). Participants • Seventy-eight patients, who had a mean duration of MSs of 46 mo, participated in the study. Intervention • After 4 wk without treatment, which functioned as a period providing an individual control, each participant took 1000 mg of PSO daily in 2 capsules for 8 wk. Outcome Measures • The symptom severity was scored on the German version of the menopausal rating scale (MRS) at baseline, after 4 wk without treatment, after 4 wk of treatment, and postintervention, with 0 = absence of symptoms and 4 = very strong symptoms. The efficacy and tolerability were estimated on scales from 0-4. Each participant's 17ß estradiol was determined at baseline and after postintervention using the patient's sera. The content of the ß-sitosterol was determined in the PSO preparations by gas chromatography. Results • The content of ß-sitosterol in the PSO used in the study was 6.3 mg/1000 mg. In the intention to treat analysis, most MRS symptoms were significantly and relevantly reduced (eg, hot flushes changed from 2.32 ± 1.04 to 1.41 ± 1.07, P < .001). Remarkably, urogenital tract symptoms (ie, a dry vagina) also significantly improved, moving from 1.32 ± 1.43 to 0.85 ± 1.19, P < .001. Few patients reported gastrointestinal symptoms. The tolerability was excellent at 3.69 ± 0.71 after 4 wk of treatment and 3.71 ± 0.65 after 8 wk of treatment. The 17ß estradiol was unchanged. Conclusions • Participants showed significant improvements in all domains of the MRS, remarkably including the difficult-to-treat urogenital symptoms. No changes occurred in the 17ß-estradiol in patients' sera after the PSO treatment. The results are promising and encourage the investigation of PSO rich in ß-sitosterol for treatment of MSs in placebo-controlled studies.

Cyclodialysis: an update.

Cyclodialysis is the result of the separation of the longitudinal ciliary muscle fibers from the scleral spur, which creates an abnormal pathway for aqueous humor drainage that may lead to ocular hypotony. For many years cyclodialysis was considered a treatment option for glaucoma. However, today it usually occurs as a complication of blunt trauma or more rarely as a complication of anterior segment ocular surgery. Ocular hypotony can lead to cataract development, optic disk swelling, refractive changes, and several retinal complications, making accurate identification and timely intervention of the cleft mandatory. Traditionally gonioscopy was the only available technique to diagnose and localize the cleft. However, other tests such as optical coherence tomography, magnetic resonance imaging, transillumination, and specially ultrasound biomicroscopy are now available for the diagnosis of cyclodialysis. Multiple treatment options are also available for this condition. Although medical treatment can be effective to close small clefts, surgery is needed in most patients to restore ocular pressure.

Branching of keratin intermediate filaments.

Keratin intermediate filaments (IFs) are crucial to maintain mechanical stability in epithelial cells. Since little is known about the network architecture that provides this stiffness and especially about branching properties of filaments, we addressed this question with different electron microscopic (EM) methods. Using EM tomography of high pressure frozen keratinocytes, we investigated the course of several filaments in a branching of a filament bundle. Moreover we found several putative bifurcations in individual filaments. To verify our observation we also visualized the keratin network in detergent extracted keratinocytes with scanning EM. Here bifurcations of individual filaments could unambiguously be identified additionally to bundle branchings. Interestingly, identical filament bifurcations were also found in purified keratin 8/18 filaments expressed in Escherichia coli which were reassembled in vitro. This excludes that an accessory protein contributes to the branch formation. Measurements of the filament cross sectional areas showed various ratios between the three bifurcation arms. This demonstrates that intermediate filament furcation is very different from actin furcation where an entire new filament is attached to an existing filament. Instead, the architecture of intermediate filament bifurcations is less predetermined and hence consistent with the general concept of IF formation.

Radical-Mediated C-H Functionalization: A Strategy for Access to Modified Cyclodextrins.

A simple and efficient radical C-H functionalization to access modified cyclodextrins (CDs) has been developed. The well-defined conformation of glycosidic and aglyconic bonds in α-, ß-, and γ-CDs favors the intramolecular 1,8-hydrogen atom transfer (HAT) promoted by the 6I-O-yl radical, which abstracts regioselectively the hydrogen at C5II of the contiguous pyranose. The C5II-radical evolves by a polar crossover mechanism to a stable 1,3,5-trioxocane ring between two adjacent glucoses or alternatively triggers the inversion of one α-d-glucose into a 5-C-acetoxy-ß-l-idose unit possessing a 1C4 conformation. The 6I,IV- and 6I,III-diols of α- and ß-CDs behave similarly to the monoalcohols, forming mostly compounds originating from two 1,8-HAT consecutive processes. In the case of 6I,II-diols the proximity of the two 6-O-yl radicals in adjacent sugar units allows the formation of unique lactone rings within the CD framework via a 1,8-HAT-ß-scission tandem mechanism. X-ray diffraction carried out on the crystalline 1,4-bis(trioxocane)-α-CD derivative shows a severe distortion toward a narrower elliptical shape for the primary face.

Natural transmission of Leishmania infantum through experimentally infected Phlebotomus perniciosus highlights the virulence of Leishmania parasites circulating in the human visceral leishmaniasis outbreak in Madrid, Spain.

A human leishmaniasis outbreak is occurring in the Madrid region, Spain, with the parasite and vector involved being Leishmania infantum and Phlebotomus perniciosus respectively. The aim of this study was to investigate the virulence of L. infantum isolates from the focus using a natural transmission model. Hamsters were infected by intraperitoneal inoculation (IP) or by bites of sand flies experimentally infected with L. infantum isolates obtained from P. perniciosus collected in the outbreak area (IPER/ES/2012/BOS1FL1 and IPER/ES/2012/POL2FL6) and a well characterized L. infantum strain JPCM5 (MCAN/ES/98/LLM-877). Hamster infections were monitored by clinical examination, serology, culture, parasite burden, Giemsa-stained imprints, PCR, histopathology and xenodiagnostic studies. Establishment of infection of L. infantum was achieved with the JPCM5 strain and outbreak isolates by both P. perniciosus infective bites or IP route. However, high virulence of BOS1FL1 and POL2FL6 isolates was highlighted by the clinical outcome of disease, high parasite detection in spleen and liver, high parasitic loads and positivity of Leishmania serology. Transmission by bite of POL2FL6 infected flies generated a slower progression of clinical disease than IP infection, but both groups were infective to P. perniciosus by xenodiagnosis at 2 months post-infection. Conversely, hamsters inoculated with JPCM5 were not infective to sand flies. Histopathology studies confirmed the wide spread of POL2FL6 parasites to several organs. A visceral leishmaniasis model that mimics the natural transmission in nature allowed us to highlight the high virulence of isolates that are circulating in the focus. These findings contribute to a better understanding of the outbreak epidemiology.

Easy access to modified cyclodextrins by an intramolecular radical approach.

A simple method to modify the primary face of cyclodextrins (CDs) is described. The 6(I)-O-yl radical of α-, ß-, and γ-CDs regioselectively abstracts the H5(II), located in the adjacent D-glucose unit, by an intramolecular 1,8-hydrogen-atom-transfer reaction through a geometrically restricted nine-membered transition state to give a stable 1,3,5-trioxocane ring. The reaction has been extended to the 1,4-diols of α- and ß-CD to give the corresponding bis(trioxocane)s. The C2-symmetric bis(trioxocane) corresponding to the α-CD is a stable crystalline solid whose structure was confirmed by X-ray diffraction analysis. The calculated geometric parameters confirm that the primary face is severely distorted toward a narrower elliptical shape for this rim.

Photochemistry of α-diketones in carbohydrates: anomalous Norrish type†II photoelimination and Norrish-Yang photocyclization promoted by the internal carbonyl group.

A series of four α-diketones placed as 1α-pyruvoyl tethers on D-glucopyranose and D-glucopyranosiduronic acid skeletons was prepared in order to determine the influence of captodative and stereoelectronic effects on the regioselectivity of the hydrogen atom transfer (HAT) in Norrish type II photochemical processes. We observed that the 1,5-HAT regioselectivity can be switched between the two potentially abstractable syn-1,3-diaxial hydrogens at H6 and H8. Highly unusual photoproducts from Norrish type II photoelimination and Norrish-Yang photocyclization initiated by the excited internal carbonyl group were obtained, in some cases in excellent synthetic yield. The 1,5-HAT transition state in the Norrish type II photoelimination was investigated by photochemical experiments in the crystalline state.

Structural and functional comparisons of salivary α-glucosidases from the mosquito vectors Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus.

Mosquito vectors of medical importance both blood and sugar feed, and their saliva contains bioactive molecules that aid in both processes. Although it has been shown that the salivary glands of several mosquito species exhibit α-glucosidase activities, the specific enzymes responsible for sugar digestion remain understudied. We therefore expressed and purified three recombinant salivary α-glucosidases from the mosquito vectors Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus and compared their functions and structures. We found that all three enzymes were expressed in the salivary glands of their respective vectors and were secreted into the saliva. The proteins, as well as mosquito salivary gland extracts, exhibited α-glucosidase activity, and the recombinant enzymes displayed preference for sucrose compared to p-nitrophenyl-α-D-glucopyranoside. Finally, we solved the crystal structure of the Ae. aegypti α-glucosidase bound to two calcium ions at a 2.3 Ångstrom resolution. Molecular docking suggested that the Ae. aegypti α-glucosidase preferred di- or polysaccharides compared to monosaccharides, consistent with enzymatic activity assays. Comparing structural models between the three species revealed a high degree of similarity, suggesting similar functional properties. We conclude that the α-glucosidases studied herein are important enzymes for sugar digestion in three mosquito species.

Mapping the risk of exposure to Crimean-Congo haemorrhagic fever virus in the Iberian Peninsula using Eurasian wild boar (Sus scrofa) as a model.

Crimean-Congo haemorrhagic fever (CCHF) virus (CCHFV) is a tick-borne zoonotic pathogen that can cause a lethal haemorrhagic disease in humans. Although the virus appears to be endemically established in the Iberian Peninsula, CCHF is an emerging disease in Spain. Clinical signs of CCHFV infection are mainly manifested in humans, but the virus replicates in several animal species. Understanding the determinants of CCHFV exposure risk from animal models is essential to predicting high-risk exposure hotspots for public health action. With this objective in mind, we designed a cross-sectional study of Eurasian wild boar (Sus scrofa) in Spain and Portugal. The study analysed 5,291 sera collected between 2006 and 2022 from 90 wild boar populations with a specific double-antigen ELISA to estimate CCHFV serum prevalence and identify the main determinants of exposure probability. To do so, we statistically modelled exposure risk with host- and environment-related predictors and spatially projected it at a 10 × 10 km square resolution at the scale of the Iberian Peninsula to map foci of infection risk. Fifty-seven (63.3 %) of the 90 populations had at least one seropositive animal, with seroprevalence ranging from 0.0 to 88.2 %. Anti-CCHFV antibodies were found in 1,026 of 5,291 wild boar (19.4 %; 95 % confidence interval: 18.3-20.5 %), with highest exposure rates in southwestern Iberia. The most relevant predictors of virus exposure risk were wild boar abundance, local rainfall regime, shrub cover, winter air temperature and soil temperature variation. The spatial projection of the best-fit model identified high-risk foci as occurring in most of western and southwestern Iberia and identified recently confirmed risk foci in eastern Spain. The results of the study demonstrate that serological surveys of CCHFV vector hosts are a powerful, robust and highly informative tool for public health authorities to take action to prevent human cases of CCHF in enzootic and emergency settings.