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Cultural heritage (CH) sites are frequently exposed to natural elements, and their exposure becomes particularly precarious with the onset of climate change. This increased vulnerability places these sites at risk of deterioration or complete destruction. Risks such as land deformation, floods, acid rain, and erosion significantly threaten historic monuments, while water-related hazards, significantly influenced by both climate change and human activities, present a particularly grave risk to these invaluable sites. Considerable research efforts have focused on safeguarding CH sites. However, there remains a deficiency in systemic approaches towards identifying and mitigating risks for CH sites. The TRIQUETRA project proposes a technological toolbox and a methodological framework for tackling climate change risks and natural hazards threatening CH in the most efficient way possible. It aims at creating an evidence-based assessment platform allowing precise risk stratification as well as a database of available mitigation measures and strategies, acting as a Decision Support System (DSS) towards efficient risk mitigation and site remediation. TRIQUETRA is a European project that brings together a diverse group of researchers with varied expertise, encompassing university research groups, research institutes, public entities, as well as small and medium-sized enterprises. In this article, TRIQUETRAs overall methodology is presented, and preliminary results concerning risk identification, TRIQUETRAs knowledge base, as well as novel sensors and coatings, are discussed.

Trimethylamine (TMA), a byproduct of gut microbiota metabolism from dietary precursors, is not only the precursor of trimethylamine-N-oxide (TMAO) but may also affect gut health. An in vitro model of intestinal epithelium of Caco-2 cells was used to evaluate the impact of TMA on inflammation, paracellular permeability, epigenetics and mitochondrial functions. The expression levels of pro-inflammatory cytokines (IL-6, IL-1β) increased significantly after 24 h exposure to TMA 1 mM. TMA exposure was associated with an upregulation of SIRT1 (TMA 1 mM, 400 μM, 10 μM) and DNMT1 (TMA 1 mM, 400 µM) genes, while DNMT3A expression decreased (TMA 1 mM). In a cell-free model, TMA (from 0.1 µM to 1 mM) induced a dose-dependent reduction in Sirtuin enzyme activity. In Caco-2 cells, TMA reduced total ATP levels and significantly downregulated ND6 expression (TMA 1 mM). TMA excess (1 mM) reduced intracellular mitochondrial DNA copy numbers and increased the methylation of the light-strand promoter in the D-loop area of mtDNA. Also, TMA (1 mM, 400 µM, 10 µM) increased the permeability of Caco-2 epithelium, as evidenced by the reduced transepithelial electrical resistance values. Based on our preliminary results, TMA excess might promote inflammation in intestinal cells and disturb epigenetic and mitochondrial homeostasis.

This paper deals with the mitigation of magnetic field levels produced by a wireless power transfer (WPT) system to recharge the battery of an electric vehicle (EV). In this work, an array of active coils surrounding the WPT coils is proposed as a mitigation technique. The theory and new methodological aspects are the focus of the paper. Magnetic field levels in the environment are calculated numerically without and with the presence of an array of active coils in a stationary WPT system for automotive applications. By the proposed mitigation method, the field levels beside the vehicle are significantly reduced and comply with the reference levels (RLs) of the ICNIRP 2010 guidelines for human exposure to electromagnetic fields and the magnetic flux density limits proposed by ISO 14117 for electromagnetic interference (EMI) in cardiac implantable electronic devices (CIEDs).

The Maltese archipelago is renowned for its spectacular coasts, characterized by vertical cliffs and scree slopes. In the western sector of Malta and the eastern region of Gozo, a marly clay formation with ductile properties underlying a stiff limestone unit has led to relevant lateral spreading. Utilizing drone aerial photogrammetry, digital elevation models, and satellite imagery, we analyzed the ongoing geomorphological processes across five promontories, selected as case studies. Our analysis reveals a complex interaction between geological structures, Quaternary sea level fluctuations, and lateral spreading processes. Photogrammetric models show that once detached, blocks from the plateaus tend to topple and fall or experience subsidence and backtilting. At Rdum il-Qammieħ, fractures up to 250 m long and openings of up to 2 m were observed, while at Sopu, detached blocks exhibit subsidence of up to 50% and rotations nearing 60°. In all the studied promontories, rotational slides predominantly occur at the frontal sectors, while toppling mechanisms are more common along scarp-edged plateaus. The thickness ratio between the stiff and the ductile formation, ranging from 0.13 to 1.12, along with slope gradients between 10° and 41°, further influence the stability of these coastal features. We discuss the structural and sea level influences on Maltese coastal cliff development over the last 125 ky. We propose a conceptual model outlining the evolution of the Malta Graben promontories through a three-stage evolutionary model: proto-promontories, cliff demolition, and isolation. This model emphasizes the significant role of predisposing, preparatory, and triggering factors in the geomorphological evolution of the Maltese coastline. Our findings provide essential insights into the landscape changes in the Maltese archipelago and represent a useful tool for coastal management and hazard mitigation strategies.

The first report of African swine fever virus (ASFV) genotype II in Italy in 2022 marked the beginning of a significant invasion in at least eight Italian regions with different infection clusters. In this study, we used the multi-gene approach to investigate the epidemiological associations between ASFV strains causing cases and outbreaks in wild boar and pigs in Italy from January 2022 to the end of 2023. Our results confirm that all the tested ASFV-positive Italian samples belonged to genotype II and show high homology with genotype II ASFV sequences previously collected in Eurasian countries. Molecular characterization revealed the presence of four genetic groups in Italy. The majority of African swine fever (ASF) samples analyzed in the current study (72%) belonged to genetic group 3, which was the most representative in Europe. The results also provide evidence of the prevalence of genetic group 19 (15.9%). In addition, we identified new putative genetic groups, genetic group 25 (9.1%) and genetic group 26 (3.0%), which have never been described before. This is the first detailed report on the molecular characterization of more than 130 ASFV strains circulating in Italy.

Background: African Swine Fever (ASF) is an infectious disease that affects domestic pig and wild boar populations. The African Swine Fever Virus (ASFV) has a genome characterized by a very complex DNA (170-193 kb) that encodes for more than 200 different proteins. Among these, the highly immunogenic phosphoprotein p30 plays a fundamental role in the induction of specific antibodies. To date, the lack of a vaccine against the disease requires continuous studies to improve knowledge about the virus and the development of new tests in addition to virological ones. Aim: The aim of this work was to produce specific monoclonal antibodies (mAbs) against the p30 protein of ASFV, which could find useful applications in routine diagnostics and the implementation of new diagnostic tools. Methods: ASFV p30 encoding gene was amplified and used for the generation of the recombinant baculovirus by transfection of the Sf21 insect cells. The recombinant protein was analyzed by immunofluorescence assay, purified, and used for mice Balb-c immunization. The hybridomas obtained were cultured and screened, using an indirect Enzyme-linked Immunosorbent Assay (iELISA), in order to select clones that secrete the mAbs of interest. Results: The expression of recombinant p30 protein was assessed using direct Immunofluorescence. The purified p30 protein fractions were analyzed by Coomassie gels staining confirming the presence of bands with a molecular weight of 30 kDa and used for the immunization of Balb-c mice. Six clones of pure hybridomas secreting the specific mAbs against recombinant p30 were obtained and tested in iELISA. The mAbs were also characterized by Western blot and Immunofluorescence assay. The best results were obtained with the anti-p30 mAb 2B8E10 clone which showed high reactivity with both recombinant and viral p30 protein, respectively. Conclusion: In this work, a recombinant p30 protein produced in an insect cell system was purified and used to immunize Balb-c mice. Six anti-p30 mAbs-secreting hybridomas clone cells were obtained. These mAbs displayed high reactivity against the recombinant protein, but only 2B8E10 mAb showed excellent functionality against the p30 protein produced by ASFV. These results open the possibility to develop different diagnostic assays. [Open Vet J 2023; 13(3.000): 358-364]

African swine fever (ASF) is a severe viral disease characterized by high lethality in suids and caused by the African Swine Fever Virus (ASFV). The ASF genotype I virus was introduced to Europe in 1957, marking the onset of the first European epidemic wave. In 2007, ASFV genotype II was detected in Georgia, affecting domestic pigs and wild boars before spreading to various European and extra-European countries, including Italy. The first case of ASFV in Italy was documented on 7 January 2022, in a wild boar in the Piedmont region. Since then, several ASFV-positive wild boar carcasses have been identified in the Piedmont and Liguria regions. By June 2023, ASFV had spread to Lombardy, one of the major pig-producing regions in northern Italy; the virus was first detected in early summer in wild boar carcasses. Two months later, it was diagnosed in a commercial pig farm as a consequence of the disease’s spread amongst wild boars and an increase in the viral environmental load. This report aims to describe the features of ASFV domestic pig outbreaks that occurred in the Zinasco municipality (Lombardy) and the joint efforts to mitigate potential direct and indirect economic impacts on the Italian and global pig industry. The epidemiological investigation and the measures implemented, which were all performed according to national and European regulations, as well as exceptional ad hoc measures aimed at protecting the pig industry, are described in order to provide a practical and effective approach to combating ASF.

African swine fever (ASF) is a disease that is a growing threat to the global swine industry. Regulations and restrictions are placed on swine movement to limit the spread of the virus. However, these are costly and time-consuming. Therefore, this study aimed to determine if high-pressure processing (HPP) sanitization techniques would be effective against the ASF virus. Here, it was hypothesized that HPP could inactivate or reduce ASF virus infectivity in tissue homogenates. To test this hypothesis, 30 aliquots of each homogenate (spleen, kidney, loin) were challenge-infected with the Turin/83 strain of ASF, at a 10 7.20 median hemadsorption dose (HAD)50/mL. Subsequently, eight aliquots of each homogenate were treated with 600 millipascal (600 MPa) HPP for 3, 5, and 7 min. Six untreated aliquots were used as the controls. Virological results showed a reduction in the viral titer of more than 7-log. These results support the validity of the study hypothesis since HPP treatment was effective in inactivating ASFV in artificially prepared samples. Overall, this study suggests the need for further investigation of other ASFV-contaminated meat products.

African swine fever (ASF) is a contagious disease of wild boar and domestic pigs notifiable to the World Organisation for Animal Health due to its high socio-economic impact. ASF is caused by the complex ASF virus (ASFV), and it can present different clinical manifestations that can be confused with other diseases; for this reason, laboratory testing is necessary for the proper diagnosis of clinically suspected animals. Despite the efforts put into it over decades, no treatment or safe vaccine is globally available, and disease control is based on early diagnosis and the implementation of strict biosecurity measures. In this context, rapid tests have the potential to accelerate and facilitate the identification of infected animals by giving fast on-site results. In this work, we improved the available point-of-care assays for the diagnosis of the disease by the development of a more specific antigen test and a more sensitive antibody test. This antibody detection test allowed for the earlier detection of infected animals than two commercial indirect ELISAs (statistically significant). Moreover, we developed a combined dual rapid test, unifying, in the same cassette, an antigen detection strip and an antibody detection strip. In this study, we confirmed that this combo approach is a useful tool for implementing rapid tests in the field since it increases the percentage of positive samples detected, even when PCR turns negative, while maintaining a good specificity.

Seronegative latent carriers (SNLCs) are animals that carry the virus without detectable antibodies and pose a risk for disease transmission and diagnostic challenges, suggesting the importance of consideration of marker vaccines in managing them. Therefore, in this study, we evaluated two modified live infectious bovine rhinotracheitis (IBR) marker vaccines (single and double deletions) for their ability to generate SNLC calves. These vaccines were administered to four groups (n = 3 in each group) of three-month-old calves in the presence or absence of passive immunity. Three hundred days after the first vaccination and after confirming the IBR seronegativity of all animals, dexamethasone was administered intravenously for five consecutive days. Only animals immunized with the modified live IBR marker vaccine (single deletion) in the absence of passive immunity exhibited a more enduring immune response than those vaccinated in the presence of passive immunity. Moreover, the administration of a modified live IBR marker vaccine (double deletion) to calves with passive immunity generated SNLC. These findings underscore the potential of live IBR marker vaccine (double-deletions) to aid serological diagnostic tools and develop vaccination protocols in achieving the desired immune response, particularly in the context of latent carrier status, offering valuable insights into optimizing vaccination strategies for effective IBR control.

Different engineering geological and geophysical investigations were performed at the Sopu promontory in the island of Gozo (Malta), involved in an impressive lateral spreading process due to the superimposition of a stiff limestone (ULC) on a ductile clay (BC). The applied techniques include: traditional geological and engineering geological surveys, unmanned aerial vehicles (UAV) survey, electrical resistivity tomography (ERT) survey, ground-penetrating radar (GPR) investigations, single-station seismic ambient noise measurements, and array seismic ambient noise measurements. The integration of the obtained results allowed us to reconstruct a subsoil model of the promontory that includes features related to the local geology of the slope and to the landslide process, as well as to define a conceptual model that describes the main evolution phases of the expansion process. The presence of back-tilted rock blocks with no features of polarization of Rayleigh waves evidenced the different failure mechanism of the rigid UCL plateau at the Sopu promontory with respect to the Selmun promontory, located in the close island of Malta, where the lateral spreading due to the same geological setting tends to produce unstable rock blocks with a toppling mechanism. This result encourages further future observations and analyses of this topic.

The African Swine Fever Virus (ASFV) is a DNA virus of the Asfarviridae family, Asfivirus genus. It is responsible for massive losses in pig populations and drastic direct and indirect economic impacts. The ever-growing handling of ASFV pathological material in laboratories, necessary for either diagnostic or research activities, requires particular attention to avoid accidental virus release from laboratories and its detrimental economic and environmental effects. Recently, the Commission Delegated Regulation (EU) 2020/689 of 17 December 2019 repealed the Commission Decision of 26 May 2003 reporting an ASF diagnostic manual (2003/422/EC) with the minimum and supplementary requirements for ASF laboratories. This decision generated a regulatory gap that has not been addressed yet. This paper aims to describe the Italian National Reference Laboratory (NRL) efforts to develop an effective and reliable biological containment tool for ASF laboratories and animal facilities. The tool consists of comprehensive and harmonized structural and procedural requirements for ASF laboratories and animal facilities that have been developed based on both current and repealed legislation, further entailing a risk assessment and internal audit as indispensable tools to design, adjust, and improve biological containment measures.

Bovine alphaherpesvirus-1 (BoAHV-1) infection is common in cattle worldwide. However, information on the spread of BoAHV-1-circulating strains in Italy remains limited. In this study, we investigated an outbreak characterized by severe respiratory symptoms in a cattle herd (n = 30) located in Central Italy. BoAHV-1 was isolated from three cattle in a cell culture, which confirmed viral infection. Next, we characterized one (16453/07 TN) of the three isolates of BoAHV-1 using whole-genome sequencing. BLASTn and phylogenetic analysis revealed a nucleotide identity >99% with all BoAHV-1 strains belonging to subtype 1.1, highlighting the genetic stability of the virus. This study reports the first full genomic characterization of a BoAHV-1 isolate in Italy, enriching our understanding of the genetic characteristics of the circulating BoAHV-1 strain in Italy.

Lv17/WB/Rie1-Δ24 was produced via illegitimate recombination mediated by low-dilution serial passage in the Cos7 cell line and isolated on PAM cell culture. The virus contains a huge ~26.4 Kb deletion in the left end of its genome. Lv17/WB/Rie1-ΔCD-ΔGL was generated via homologous recombination, crossing two ASFV strains (Lv17/WB/Rie1-ΔCD and Lv17/WB/Rie1-ΔGL containing eGFP and mCherry markers) during PAM co-infection. The presence of unique parental markers in the Lv17/WB/Rie1-ΔCD-ΔGL genome indicates at least two recombination events during the crossing, suggesting that homologous recombination is a relatively frequent event in the ASFV genome during replication in PAM. Pigs infected with Lv17/WB/Rie1-Δ24 and Lv17/WB/Rie1/ΔCD-ΔGL strains have shown mild clinical signs despite that ASFV could not be detected in their sera until a challenge infection with the Armenia/07 ASFV strain. The two viruses were not able to induce protective immunity in pigs against a virulent Armenia/07 challenge.

Fungicides are usually applied on strawberries to manage gray mold, induced by the fungal pathogen Botrytis cinerea. In this study, four reduced-risk fungicides (formulations of pyrimethanil, PYR, 175 mL/hL; boscalid, BOS, 80 g/hL; combination fludioxonil, FLU, +cyprodinil, CYP, 110 g/hL) were applied before harvest for the management of post-harvest diseases of strawberries. The resulting fungicide residues on the strawberry fruit were also quantified. Strawberry fruits were harvested at 0, 4, 8, and 12 days following treatment (dft) and kept at 20 ± 1 °C for 4 days or cold-stored for 7 days at 0.5 ± 1 °C, followed by a 4-day shelf life at 20 ± 1 °C. All fungicides significantly reduced gray mold, according to the McKinney Index. At 0 dft and 4 days of shelf life, the FLU + CYP completely prevented post-harvest strawberry gray mold, while PYR and BOS reduced the disease by 88% and 42%, respectively, in comparison to the untreated control. For the duration of experiment, fungicide residues were always below the maximum residue levels, and FLU was the most degraded, thanks to the enzymatic pool of the strawberries. Monitoring fungicide residues in strawberries is essential to provide the consumer information on the safety of this widely consumed fruit. The present study points out the safety of strawberry fruits for consumers, even if the treatment strategy implies the use of fungicide mixtures before the consumption, with fungicide levels always being below the MRL.

Although the COVID-19 pandemic appears to be diminishing, the emergence of SARS-CoV-2 variants represents a threat to humans due to their inherent transmissibility, immunological evasion, virulence, and invulnerability to existing therapies. The COVID-19 pandemic affected more than 500 million people and caused over 6 million deaths. Vaccines are essential, but in circumstances in which vaccination is not accessible or in individuals with compromised immune systems, drugs can provide additional protection. Targeting host signaling pathways is recommended due to their genomic stability and resistance barriers. Moreover, targeting host factors allows us to develop compounds that are effective against different viral variants as well as against newly emerging virus strains. In recent years, the globe has experienced climate change, which may contribute to the emergence and spread of infectious diseases through a variety of factors. Warmer temperatures and changing precipitation patterns can increase the geographic range of disease-carrying vectors, increasing the risk of diseases spreading to new areas. Climate change may also affect vector behavior, leading to a longer breeding season and more breeding sites for disease vectors. Climate change may also disrupt ecosystems, bringing humans closer to wildlife that transmits zoonotic diseases. All the above factors may accelerate the emergence of new viral epidemics. Plant-derived products, which have been used in traditional medicine for treating pathological conditions, offer structurally novel therapeutic compounds, including those with anti-viral activity. In addition, plant-derived bioactive substances might serve as the ideal basis for developing sustainable/efficient/cost-effective anti-viral alternatives. Interest in herbal antiviral products has increased. More than 50% of approved drugs originate from herbal sources. Plant-derived compounds offer diverse structures and bioactive molecules that are candidates for new drug development. Combining these therapies with conventional drugs could improve patient outcomes. Epigenetics modifications in the genome can affect gene expression without altering DNA sequences. Host cells can use epigenetic gene regulation as a mechanism to silence incoming viral DNA molecules, while viruses recruit cellular epitranscriptomic (covalent modifications of RNAs) modifiers to increase the translational efficiency and transcript stability of viral transcripts to enhance viral gene expression and replication. Moreover, viruses manipulate host cells’ epigenetic machinery to ensure productive viral infections. Environmental factors, such as natural products, may influence epigenetic modifications. In this review, we explore the potential of plant-derived substances as epigenetic modifiers for broad-spectrum anti-viral activity, reviewing their modulation processes and anti-viral effects on DNA and RNA viruses, as well as addressing future research objectives in this rapidly emerging field.

Abstract Background African Swine Fever (ASF) is a challenge for pig health worldwide. The disease has spread to multiple countries on five continents. ASF‐free countries need to apply effective strategies to prevent the introduction of infection. Methods Italy implemented a surveillance and prevention plan for ASF in 2020, supported by a dedicated information system. Several pillars for action have been identified: passive surveillance in both domestic pigs and wild boar populations, verification of the implementation of biosecurity measures on farms and an awareness campaign for all involved stakeholders. Results There were some regional differences in the management of passive surveillance. In order to identify all critical points and apply corrective measures, regional authorities were called to carry out a gap analysis exercise in July 2020. There were an adequate number of samples collected from wild boar but the number of samples collected from domestic pigs was below the target in most regions. Furthermore, sample distribution within the country was not homogeneous. Conclusions During the forthcoming year, some issues must be addressed in order to establish an effective early detection system in Italian ASF‐free regions.

Trimethylamine (TMA), a byproduct of gut microbiota metabolism from dietary precursors, is not only the precursor of trimethylamine-N-oxide (TMAO) but may also affect gut health. An in vitro model of intestinal epithelium of Caco-2 cells was used to evaluate the impact of TMA on inflammation, paracellular permeability, epigenetics and mitochondrial functions. The expression levels of pro-inflammatory cytokines (IL-6, IL-1β) increased significantly after 24 h exposure to TMA 1 mM. TMA exposure was associated with an upregulation of SIRT1 (TMA 1 mM, 400 μM, 10 μM) and DNMT1 (TMA 1 mM, 400 µM) genes, while DNMT3A expression decreased (TMA 1 mM). In a cell-free model, TMA (from 0.1 µM to 1 mM) induced a dose-dependent reduction in Sirtuin enzyme activity. In Caco-2 cells, TMA reduced total ATP levels and significantly downregulated ND6 expression (TMA 1 mM). TMA excess (1 mM) reduced intracellular mitochondrial DNA copy numbers and increased the methylation of the light-strand promoter in the D-loop area of mtDNA. Also, TMA (1 mM, 400 µM, 10 µM) increased the permeability of Caco-2 epithelium, as evidenced by the reduced transepithelial electrical resistance values. Based on our preliminary results, TMA excess might promote inflammation in intestinal cells and disturb epigenetic and mitochondrial homeostasis. [ABSTRACT FROM AUTHOR]

This paper deals with the mitigation of magnetic field levels produced by a wireless power transfer (WPT) system to recharge the battery of an electric vehicle (EV). In this work, an array of active coils surrounding the WPT coils is proposed as a mitigation technique. The theory and new methodological aspects are the focus of the paper. Magnetic field levels in the environment are calculated numerically without and with the presence of an array of active coils in a stationary WPT system for automotive applications. By the proposed mitigation method, the field levels beside the vehicle are significantly reduced and comply with the reference levels (RLs) of the ICNIRP 2010 guidelines for human exposure to electromagnetic fields and the magnetic flux density limits proposed by ISO 14117 for electromagnetic interference (EMI) in cardiac implantable electronic devices (CIEDs). [ABSTRACT FROM AUTHOR]

The Maltese archipelago is renowned for its spectacular coasts, characterized by vertical cliffs and scree slopes. In the western sector of Malta and the eastern region of Gozo, a marly clay formation with ductile properties underlying a stiff limestone unit has led to relevant lateral spreading. Utilizing drone aerial photogrammetry, digital elevation models, and satellite imagery, we analyzed the ongoing geomorphological processes across five promontories, selected as case studies. Our analysis reveals a complex interaction between geological structures, Quaternary sea level fluctuations, and lateral spreading processes. Photogrammetric models show that once detached, blocks from the plateaus tend to topple and fall or experience subsidence and backtilting. At Rdum il-Qammieħ, fractures up to 250 m long and openings of up to 2 m were observed, while at Sopu, detached blocks exhibit subsidence of up to 50% and rotations nearing 60°. In all the studied promontories, rotational slides predominantly occur at the frontal sectors, while toppling mechanisms are more common along scarp-edged plateaus. The thickness ratio between the stiff and the ductile formation, ranging from 0.13 to 1.12, along with slope gradients between 10° and 41°, further influence the stability of these coastal features. We discuss the structural and sea level influences on Maltese coastal cliff development over the last 125 ky. We propose a conceptual model outlining the evolution of the Malta Graben promontories through a three-stage evolutionary model: proto-promontories, cliff demolition, and isolation. This model emphasizes the significant role of predisposing, preparatory, and triggering factors in the geomorphological evolution of the Maltese coastline. Our findings provide essential insights into the landscape changes in the Maltese archipelago and represent a useful tool for coastal management and hazard mitigation strategies. [ABSTRACT FROM AUTHOR]

The first report of African swine fever virus (ASFV) genotype II in Italy in 2022 marked the beginning of a significant invasion in at least eight Italian regions with different infection clusters. In this study, we used the multi-gene approach to investigate the epidemiological associations between ASFV strains causing cases and outbreaks in wild boar and pigs in Italy from January 2022 to the end of 2023. Our results confirm that all the tested ASFV-positive Italian samples belonged to genotype II and show high homology with genotype II ASFV sequences previously collected in Eurasian countries. Molecular characterization revealed the presence of four genetic groups in Italy. The majority of African swine fever (ASF) samples analyzed in the current study (72%) belonged to genetic group 3, which was the most representative in Europe. The results also provide evidence of the prevalence of genetic group 19 (15.9%). In addition, we identified new putative genetic groups, genetic group 25 (9.1%) and genetic group 26 (3.0%), which have never been described before. This is the first detailed report on the molecular characterization of more than 130 ASFV strains circulating in Italy. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) is a severe viral disease of domestic pigs and Eurasian wild boars (Sus scrofa) caused by the African swine fever virus (ASFV). ASF is endemic in sub-Saharan Africa, where 24 genotypes of the virus have been reported. Between the late 1950s and the early 1980s, genotype I ASFV emerged in Europe, including Italy. In June 2007, a second ASF epidemic wave caused by genotype II was registered, involving several European and extra-European countries, including Italy in 2022. The present paper aims to provide the state of the art of ASF in Italy, describing the course of ASF in wild boars and domestic pigs as an example of multiple concurring different scenarios. Sardinia is coping with the last phase of the eradication of the disease by applying the exit strategy. Conversely, four clusters of infection located in North, Central, and South Italy are still ongoing. The unique and complex Italian experience in ASF-controlling may be useful to increase know-how on the efficacy of strategies and measures, as well as issues that could be further improved.

IntroductionAfrican swine fever (ASF) is a contagious viral disease of pigs and wild boar that poses a major threat to the global swine industry. The genotype II African swine fever virus (ASFV) entered the European Union (EU) in 2014 and since then fourteen countries have been affected, Italy and North Macedonia being the last in 2022. While whole genome sequencing remains the gold standard for the identification of new genetic markers, sequencing of multiple loci with significant variations could be used as a rapid and cost-effective alternative to track outbreaks and study disease evolution in endemic areas.Materials and methodsTo further our understanding of the epidemiology and spread of ASFV in Europe, 382 isolates collected during 2007 to 2022 were sequenced. The study was initially performed by sequencing the central variable region (CVR), the intergenic region (IGR) between the I73R and I329L genes and the O174L and K145R genes. For further discrimination, two new PCRs were designed to amplify the IGR between the 9R and 10R genes of the multigene family 505 (MGF505) and the IGR between the I329L and I215L genes. The sequences obtained were compared with genotype II isolates from Europe and Asia.ResultsThe combination of the results obtained by sequencing these variable regions allowed to differentiate the European II-ASFV genotypes into 24 different groups. In addition, the SNP identified in the IGR I329L-I215L region, not previously described, grouped the viruses from North Macedonia that caused the 2022 outbreaks with viruses from Romania, Bulgaria, Serbia and Greece, differentiating from other genotype II isolates present in Europe and Asia. Furthermore, tandem repeat sequence (TRS) within the 9R-10R genes of the multigene family 505 (MGF505) revealed eight different variants circulating.DiscussionThese findings describe a new multi-gene approach sequencing method that can be used in routine genotyping to determine the origin of new introductions in ASF-free areas and track infection dynamics in endemic areas.

ABSTRACT Traditional studies on the evolution of antibiotic resistance development use approaches that can range from laboratory-based experimental studies, to epidemiological surveillance, to sequencing of clinical isolates. However, evolutionary trajectories also depend on the environment in which selection takes place, compelling the need to more deeply investigate the impact of environmental complexities and their dynamics over time. Herein, we explored the within-patient adaptive long-term evolution of a Pseudomonas aeruginosa hypermutator lineage in the airways of a cystic fibrosis (CF) patient by performing a chronological tracking of mutations that occurred in different subpopulations; our results demonstrated parallel evolution events in the chromosomally encoded class C β-lactamase (blaPDC). These multiple mutations within blaPDC shaped diverse coexisting alleles, whose frequency dynamics responded to the changing antibiotic selective pressures for more than 26 years of chronic infection. Importantly, the combination of the cumulative mutations in blaPDC provided structural and functional protein changes that resulted in a continuous enhancement of its catalytic efficiency and high level of cephalosporin resistance. This evolution was linked to the persistent treatment with ceftazidime, which we demonstrated selected for variants with robust catalytic activity against this expanded-spectrum cephalosporin. A “gain of function” of collateral resistance toward ceftolozane, a more recently introduced cephalosporin that was not prescribed to this patient, was also observed, and the biochemical basis of this cross-resistance phenomenon was elucidated. This work unveils the evolutionary trajectories paved by bacteria toward a multidrug-resistant phenotype, driven by decades of antibiotic treatment in the natural CF environmental setting. IMPORTANCE Antibiotics are becoming increasingly ineffective to treat bacterial infections. It has been consequently predicted that infectious diseases will become the biggest challenge to human health in the near future. Pseudomonas aeruginosa is considered a paradigm in antimicrobial resistance as it exploits intrinsic and acquired resistance mechanisms to resist virtually all antibiotics known. AmpC β-lactamase is the main mechanism driving resistance in this notorious pathogen to β-lactams, one of the most widely used classes of antibiotics for cystic fibrosis infections. Here, we focus on the β-lactamase gene as a model resistance determinant and unveil the trajectory P. aeruginosa undertakes on the path toward a multidrug-resistant phenotype during the course of two and a half decades of chronic infection in the airways of a cystic fibrosis patient. Integrating genetic and biochemical studies in the natural environment where evolution occurs, we provide a unique perspective on this challenging landscape, addressing fundamental molecular mechanisms of resistance.

African swine fever virus (ASFV) is the etiological agent of a haemorrhagic disease that threatens the global pig industry. There is an urgency to develop a safe and efficient vaccine, but the knowledge of the immune–pathogenetic mechanisms behind ASFV infection is still very limited. In this paper, we evaluated the haematological and immunological parameters of domestic pigs vaccinated with the ASFV Lv17/WB/Rie1 strain or its derived mutant Lv17/WB/Rie1/d110-11L and then challenged with virulent Armenia/07 ASFV. Circulating levels of C-reactive protein (CRP), 13 key cytokines and 11 haematological parameters were evaluated throughout the study. Lv17/WB/Rie1 triggered an inflammatory response, with increased levels of CRP and pro-inflammatory cytokines, and induced lymphopenia, thrombocytopenia and a decline in red blood cell (RBC) parameters, although this was transitory. Lv17/WB/Rie1/d110-11L triggered only transitory thrombocytopenia and a mild inflammatory reaction, with no increase in serum levels of pro-inflammatory cytokines, but it raised IL-1Ra levels. Both strains counteracted several adverse reactions elicited by virulent challenge, like thrombocytopenia, a decline in RBC parameters, and inflammation. Within this paper, we provided a deep portrayal of the impact of diverse ASFV strains on the domestic pig’s immune system. A better understanding of these immune–pathological mechanisms would help to design suitable vaccines against this disease.

The genetically related assemblages of the intestinal protozoa parasite Giardia lamblia are morphologically indistinguishable and are often derived from specific hosts. The Giardia assemblages are separated by large genetic distances, which might account for their relevant biological and pathogenic differences. In this work, we analyzed the RNAs cargo released into exosomal-like vesicles (ElVs) by the assemblages A and B, which differentially infect humans, and the assemblage E, which infects hoofed animals. The RNA sequencing analysis revealed that the ElVs of each assemblage contained distinct small RNA (sRNA) biotypes, suggesting a preference for specific packaging in each assemblage. These sRNAs were classified into three categories, ribosomal-small RNAs (rsRNAs), messenger-small RNAs (msRNAs), and transfer-small RNAs (tsRNAs), which may play a regulatory role in parasite communication and contribute to host-specificity and pathogenesis. Uptake experiments showed, for the first time, that ElVs were successfully internalized by the parasite trophozoites. Furthermore, we observed that the sRNAs contained inside these ElVs were first located below the plasma membrane but then distributed along the cytoplasm. Overall, the study provides new insights into the molecular mechanisms underlying the host-specificity and pathogenesis of G. lamblia and highlights the potential role of sRNAs in parasite communication and regulation.