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Background Epizootic haemorrhagic disease (EHD) is a vector-borne viral disease of domestic and wild ruminants. Epizootic haemorrhagic disease virus (EHDV) is transmitted by Culicoides spp. EHDV is a member of the Orbivirus genus within the Reoviridae family. It shares many morphological and structural characteristics with other members of the genus, such as the bluetongue virus, African horse sickness virus, and equine encephalosis virus. Aims The purpose of our study was to investigate the epidemiological situation of EHDV in Libya in order to gain some knowledge about the presence of this virus in the country. Methods In this study, we investigated the seroprevalence of EHDV in Libya, testing 855 blood samples collected during 2015. The samples were collected from domestic ruminants (cattle, sheep, and goats) originating from 11 provinces of Libya. Sera were tested by competitive enzyme-linked immunosorbent assays and positive samples confirmed by serum neutralization test. Results The overall seroprevalence of EHDV was estimated to be 4% (95% confidence intervals = 2.8%-5.4%). Small ruminant seroprevalence was significantly (p = 0.016) higher than that found in cattle. Neutralizing antibodies against EHDV-6 were detected in a sheep from the western region of Libya. Conclusion This study suggests that EHDV has circulated or is circulating in Libya, and sheep could play an important role in the epidemiology of EHDV, and the virus may still be circulating in North Africa.

• Following the announcement on December 2020 about the emergence of a new variant (VOC 202012/01, B.1.1.7 lineage) in the United Kingdom, a targeted surveillance was put in place in the Abruzzo region (Italy), which allowed detection of 313 persons affected by lineage B.1.1.7, up to the 20th of February 2021. We investigated the results of RT-PCR on nasopharyngeal swabs tested from December 2020 to February 2021 to verify any difference on the viral load and persistence between people infected by lineage B.1.1.7 and others. Statistically significant lower values of CT associated with the detection of the N protein encoding gene (CT N) were observed in persons with lineage B.1.1.7 infection (median CT N = 15.8) in comparison to those infected by other lineages (median CT N = 16.9). A significantly longer duration of the persistence of SARS-CoV-2 RNA in nasopharyngeal swabs was observed in persons with lineage B.1.1.7 infection (16 days) in comparison to those infected by other lineages (14 days).

In a companion paper by Cohen-Adad et al. we introduce the spine generic quantitative MRI protocol that provides valuable metrics for assessing spinal cord macrostructural and microstructural integrity. This protocol was used to acquire a single subject dataset across 19 centers and a multi-subject dataset across 42 centers (for a total of 260 participants), spanning the three main MRI manufacturers: GE, Philips and Siemens. Both datasets are publicly available via git-annex. Data were analysed using the Spinal Cord Toolbox to produce normative values as well as inter/intra-site and inter/intra-manufacturer statistics. Reproducibility for the spine generic protocol was high across sites and manufacturers, with an average inter-site coefficient of variation of less than 5% for all the metrics. Full documentation and results can be found at https://spine-generic.rtfd.io/. The datasets and analysis pipeline will help pave the way towards accessible and reproducible quantitative MRI in the spinal cord., Measurement(s)spinal cordTechnology Type(s)magnetic resonance imagingFactor Type(s)manufacturer • siteSample Characteristic - OrganismHomo sapiensSample Characteristic - LocationCanada • Switzerland • Australia • United States of America • United Kingdom • Germany • French Republic • Czech Republic • Italy • Japan • Kingdom of Spain • China Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.14052269

West Nile Virus (WNV) is a mosquito-borne virus enzootically maintained in birds. However, it can incidentally infect other species, leading to sometimes severe clinical consequences like in horses and especially human beings. Despite the topic relevance, the presence and distribution of WNV are currently unknown in Namibia. Several countries implement surveillance systems based on virus detection in birds, mosquitoes, and vertebrate species including horses. The present study aimed to fill this knowledge gap by serologically evaluating WNV exposure in Namibian donkeys, whose population is remarkably bigger than the horse one. Forty-seven out of 260 sampled animals showed neutralizing antibodies against WNV (18.07% [95% CI = 13.59–23.30%]), demonstrating its circulation in all country territory, although, with apparent regional differences. On the contrary, no association with animal age or sex could be identified. The present study demonstrates the widespread presence of WNV in Namibia as well as the practical utility and effectiveness of donkeys as sentinels for infection surveillance. Due to clinical relevance, vaccination campaigns should be considered for horses of high economic or genetic value. Additionally, the burden of WNV infection on human health should be carefully evaluated.

Objective In this study we address the automatic segmentation of selected muscles of the thigh and leg through a supervised deep learning approach. Material and methods The application of quantitative imaging in neuromuscular diseases requires the availability of regions of interest (ROI) drawn on muscles to extract quantitative parameters. Up to now, manual drawing of ROIs has been considered the gold standard in clinical studies, with no clear and universally accepted standardized procedure for segmentation. Several automatic methods, based mainly on machine learning and deep learning algorithms, have recently been proposed to discriminate between skeletal muscle, bone, subcutaneous and intermuscular adipose tissue. We develop a supervised deep learning approach based on a unified framework for ROI segmentation. Results The proposed network generates segmentation maps with high accuracy, consisting in Dice Scores ranging from 0.89 to 0.95, with respect to “ground truth” manually segmented labelled images, also showing high average performance in both mild and severe cases of disease involvement (i.e. entity of fatty replacement). Discussion The presented results are promising and potentially translatable to different skeletal muscle groups and other MRI sequences with different contrast and resolution.

From 16 March to 15 December 2020, 132,357 naso-pharyngeal/oropharyngeal swabs were collected in the province of Teramo, Abruzzo Region, Italy, and tested for the presence of SARS-CoV-2 genomic RNA by a commercially available molecular assay. A total of 12,880 swabs resulted positive. For 8212 positive patients (4.150 women and 4.062 men) the median age was statistically different between women (median: 49.55 ± 23.9 of SD) and men (median: 48.35 ± 23.5 of SD) while no differences were found in the comparison between the cycle threshold for the N protein-encoding gene (CT N) median values and gender. Differences were observed in the CT N gene median values of swabs collected from March to September as well as in the pairwise comparison between September and October and between November and December. The CT N gene median values observed in specific periods characterizing the SARS-CoV-2 epidemic in 2020 were also compared with the incidence of COVID-19 cases; a strong inverse correlation was highlighted (Pearson correlation coefficient = −0.978). Our findings confirm the usefulness of the CT N values as an indirect detection parameter to monitor viral loads in the population.

Deep gray matter nuclei are the synaptic relays, responsible to route signals between specific brain areas. Dentate nuclei (DNs) represent the main output channel of the cerebellum and yet are often unexplored especially in humans. We developed a multimodal MRI approach to identify DNs topography on the basis of their connectivity as well as their microstructural features. Based on results, we defined DN parcellations deputed to motor and to higher‐order functions in humans in vivo. Whole‐brain probabilistic tractography was performed on 25 healthy subjects from the Human Connectome Project to infer DN parcellations based on their connectivity with either the cerebral or the cerebellar cortex, in turn. A third DN atlas was created inputting microstructural diffusion‐derived metrics in an unsupervised fuzzy c‐means classification algorithm. All analyses were performed in native space, with probability atlas maps generated in standard space. Cerebellar lobule‐specific connectivity identified one motor parcellation, accounting for about 30% of the DN volume, and two non‐motor parcellations, one cognitive and one sensory, which occupied the remaining volume. The other two approaches provided overlapping results in terms of geometrical distribution with those identified with cerebellar lobule‐specific connectivity, although with some differences in volumes. A gender effect was observed with respect to motor areas and higher‐order function representations. This is the first study that indicates that more than half of the DN volumes is involved in non‐motor functions and that connectivity‐based and microstructure‐based atlases provide complementary information. These results represent a step‐ahead for the interpretation of pathological conditions involving cerebro‐cerebellar circuits., We developed a multimodal MRI approach to identify DNs topography on the basis of their connectivity, in turn with either the cerebral or the cerebellar cortex, as well as their microstructural features, inputting them in an unsupervised fuzzy c‐means classification algorithm. The three approaches provided quite overlapping results in terms of geometrical distribution and, for the first time, indicate that more than half of the DN volumes is involved in non‐motor higher‐order functions.

Introduction: Nusinersen is a recent promising therapy approved for the treatment of spinal muscular atrophy (SMA), a rare disease characterized by the degeneration of alpha motor neurons (αMN) in the spinal cord (SC) leading to progressive muscle atrophy and dysfunction. Muscle and cervical SC quantitative magnetic resonance imaging (qMRI) has never been used to monitor drug treatment in SMA. The aim of this pilot study is to investigate whether qMRI can provide useful biomarkers for monitoring treatment efficacy in SMA.Methods: Three adult SMA 3a patients under treatment with nusinersen underwent longitudinal clinical and qMRI examinations every 4 months from baseline to 21-month follow-up. The qMRI protocol aimed to quantify thigh muscle fat fraction (FF) and water-T2 (w-T2) and to characterize SC volumes and microstructure. Eleven healthy controls underwent the same SC protocol (single time point). We evaluated clinical and imaging outcomes of SMA patients longitudinally and compared SC data between groups transversally.Results: Patient motor function was stable, with only Patient 2 showing moderate improvements. Average muscle FF was already high at baseline (50%) and progressed over time (57%). w-T2 was also slightly higher than previously published data at baseline and slightly decreased over time. Cross-sectional area of the whole SC, gray matter (GM), and ventral horns (VHs) of Patients 1 and 3 were reduced compared to controls and remained stable over time, while GM and VHs areas of Patient 2 slightly increased. We found altered diffusion and magnetization transfer parameters in SC structures of SMA patients compared to controls, thus suggesting changes in tissue microstructure and myelin content.Conclusion: In this pilot study, we found a progression of FF in thigh muscles of SMA 3a patients during nusinersen therapy and a concurrent slight reduction of w-T2 over time. The SC qMRI analysis confirmed previous imaging and histopathological studies suggesting degeneration of αMN of the VHs, resulting in GM atrophy and demyelination. Our longitudinal data suggest that qMRI could represent a feasible technique for capturing microstructural changes induced by SMA in vivo and a candidate methodology for monitoring the effects of treatment, once replicated on a larger cohort.

Novel SARS-CoV-2 variants with potential impacts on diagnostics, antivirals, and vaccines are spreading in Italy. In this editorial, we highlight the role that veterinary public health institutes may have in this global crisis, as their expertise in genomic/antigenic surveillance and animal studies are crucial to tackle SARS-CoV-2 pandemic.

Highlights • Replication and tropism of SARS-CoV-2 in cattle, sheep, and pigs using EVOCs, were investigated. • Respiratory tissues of cattle and sheep, but not those of pigs, are able to sustain viral replication. • A SARS-CoV-2 isolate harbouring mutation D614 G in the S protein has greater replication capabilities. • SARS-CoV-2 binds to ACE2-expressing cells of the respiratory tract of cattle and sheep., There is strong evidence that severe acute respiratory syndrome 2 virus (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, originated from an animal reservoir. However, the exact mechanisms of emergence, the host species involved, and the risk to domestic and agricultural animals are largely unknown. Some domestic animal species, including cats, ferrets, and minks, have been demonstrated to be susceptible to SARS-CoV-2 infection, while others, such as pigs and chickens, are not. Importantly, the susceptibility of ruminants to SARS-CoV-2 is unknown, even though they often live in close proximity to humans. We investigated the replication and tissue tropism of two different SARS-CoV-2 isolates in the respiratory tract of three farm animal species - cattle, sheep, and pigs - using respiratory ex vivo organ cultures (EVOCs). We demonstrate that the respiratory tissues of cattle and sheep, but not of pigs, sustain viral replication in vitro of both isolates and that SARS-CoV-2 is associated to ACE2-expressing cells of the respiratory tract of both ruminant species. Intriguingly, a SARS-CoV-2 isolate containing an amino acid substitution at site 614 of the spike protein (mutation D614G) replicated at higher magnitude in ex vivo tissues of both ruminant species, supporting previous results obtained using human cells. These results suggest that additional in vivo experiments involving several ruminant species are warranted to determine their potential role in the epidemiology of this virus.

In a companion paper by Cohen-Adad et al. we introduce the spine generic quantitative MRI protocol that provides valuable metrics for assessing spinal cord macrostructural and microstructural integrity. This protocol was used to acquire a single subject dataset across 19 centers and a multi-subject dataset across 42 centers (for a total of 260 participants), spanning the three main MRI manufacturers: GE, Philips and Siemens. Both datasets are publicly available via git-annex. Data were analysed using the Spinal Cord Toolbox to produce normative values as well as inter/intra-site and inter/intra-manufacturer statistics. Reproducibility for the spine generic protocol was high across sites and manufacturers, with an average inter-site coefficient of variation of less than 5% for all the metrics. Full documentation and results can be found at https://spine-generic.rtfd.io/ . The datasets and analysis pipeline will help pave the way towards accessible and reproducible quantitative MRI in the spinal cord.

The current pandemic is caused by a novel coronavirus (CoV) called SARS-CoV-2 (species Severe acute respiratory syndrome-related coronavirus, subgenus Sarbecovirus, genus Betacoronavirus, family Coronaviridae). In Italy, up to the 2nd of April 2020, overall 139,422 confirmed cases and 17,669 deaths have been notified, while 26,491 people have recovered. Besides the overloading of hospitals, another issue to face was the capacity to perform thousands of tests per day. In this perspective, to support the National Health Care System and to minimize the impact of this rapidly spreading virus, the Italian Ministry of Health involved the Istituti Zooprofilattici Sperimentali (IZSs), Veterinary Public Health Institutes, in the diagnosis of SARS-CoV-2 by testing human samples. The Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise is currently testing more than 600 samples per day and performing whole genome sequencing from positive samples. Sequence analysis of these samples suggested that different viral variants may be circulating in Italy, and so in Abruzzo region. CoVs, and related diseases, are well known to veterinarians since decades. The experience that veterinarians operating within the Public Health system gained in the control and characterization of previous health issues of livestock and poultry including avian flu, bluetongue, foot and mouth disease, responsible for huge economic losses, is certainly of great help to minimize the impact of this global crisis., Highlights • Italian VPH institutes (IZSs) are currently involved in SARS-CoV-2 diagnosis. • IZSAM is processing 600 samples from Abruzzo region per day. • 16 complete sequences were deposited on GISAID. • Outbreaks in Abruzzo are related with those from northern Italy and northern Europe. • Veterinarians play a crucial role in the diagnosis and characterization of SARS-CoV-2.

Since February 2020, Italy has been seriously affected by the SARS-CoV-2 pandemic. To support the National Health Care system, naso-pharyngeal/oropharyngeal swabs collected from suspected cases of Teramo province, Abruzzo region, are tested at Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise G. Caporale, for the presence of SARS-CoV-2 RNA. Out of 12,446 tested individuals, 605 returned positive results at least once, with prevalence significantly higher in men. A reduction of the level of viral RNA in the first swab per each positive patient collected over time was also observed. Moreover, 81 patients had at least one positive sample and two final negative tests: positivity in swabs lasted from 14 to 63 days, with a median value of 30 days. This shows the potential for the virus to coexist with patients for a long time, although we highlighted intermittent positivity in several cases. The evolution of the SARS-CoV-2 epidemiological situation and knowledge on viral shedding should be closely monitored, to interpret the findings correctly and adjust accordingly the surveillance activities.

This study reports the monitoring of several emerging viral pathogens in Mauritania, which was carried out by the analysis of bovine and camel samples taken at the slaughterhouse of Nouakchott. Blood and serum were collected by random sampling from 159 camels and 118 cattle in March 2013 at the large animals abattoir in Nouakchott. Serological tests for Rift Valley Fever (RVF), Peste des Petits Ruminants (PPR), West Nile disease (WND), epizootic haemorrhagic disease (EHD) and African horse sickness (AHS) were carried out using commercial ELISA kits. The samples, which resulted positives for PPR, WND and AHS, were tested with the confirmatory virus neutralization test (VNT). According to ELISA results, serological prevalence of RVF was 45% (95% CI 52.3-37.7) in camels and 16% (95% CI 22.6-9.4) in cattle. The difference between the observed prevalences in camels and in cattle was significant (p value ≤ 0.01). PPR was absent in camels and had 12% prevalence (95% CI, 17.86-6.14) in cattle. Furthermore, camels showed 92% (95% CI, 96.1-87.9) prevalence of WNV, 73% (95% CI, 82.3-63.64) of EHD and 3% (95% CI, 5.6-0.4) of AHS. This data are of relevance since provided useful feedbacks on the circulation of the pathogens in field. Moreover, this survey provided new information on the susceptibility of camels to several emerging pathogens and on the possible use of this species as sentinel animal.

Feline morbillivirus (FeMV) is a newly discovered paramyxovirus infecting domestic cats and its role in the pathogenesis of feline chronic kidney disease (CKD) has been suggested, however not confirmed. The primary aim of the study was to evaluate the renal damage associated with FeMV infection in cats. In this retrospective study, clinical and clinicopathological data were compared among 14 FeMV naturally infected, 21 CKD and 22 healthy cats. FeMV positive cats had serum chemistry analytes and main urine chemistry results similar to the healthy subjects. FeMV positive cats had significantly decreased urine specific gravity (median 1054, range 1022&ndash, 1065) and urine creatinine (median 227.23 mg/dL, range 83.02&ndash, 489.75) when compared with healthy cats (median 1067, range 1040&ndash, 1080, P &lt, 0.001, median 406.50 mg/dL, range 195.32&ndash, 575.58, P &lt, 0.001, respectively). Urine protein:creatinine ratio (UPC) results of FeMV and CKD were not different (median 0.20, range 0.08&ndash, 1.03, median 0.23, range 0.10&ndash, 0.80, respectively), however UPC results were significantly increased in both groups, if compared with healthy cats (median 0.1, range 0.04&ndash, 0.250, P &lt, 0.01). Based on clinical data, serum creatinine concentration, urine specific gravity and UPC results, CKD was suspected by clinicians in 3/14 FeMV cats. Urine protein sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 10/13 (77%) FeMV cats indicated a tubular pattern, with a decrease of uromodulin and an increase in the number and intensity of low molecular weight proteins. FeMV infection can be associated with different grades of renal dysfunction ranging from mild tubular proteinuria with less concentrated urine to azotemia in cats younger than those typically affected by CKD.

Usutu virus (USUV) is an emerging arbovirus isolated in 1959 (Usutu River, Swaziland). Previously restricted to sub-Saharan Africa, the virus was introduced in Europe in 1996. While the USUV has received little attention in Africa, the virus emergence has prompted numerous studies with robust epidemiological surveillance programs in Europe. The natural transmission cycle of USUV involves mosquitoes (vectors) and birds (amplifying hosts) with humans and other mammals considered incidental (“dead-end”) hosts. In Africa, the virus was isolated in mosquitoes, rodents and birds and serologically detected in horses and dogs. In Europe, USUV was detected in bats, whereas antibodies were found in different animal species (horses, dogs, squirrels, wild boar, deer and lizards). While bird mortalities were not reported in Africa, in Europe USUV was shown to be highly pathogenic for several bird species, especially blackbirds (Turdus merula) and great gray owls (Strix nebulosa). Furthermore, neurotropism of USUV for humans was reported for the first time in both immunocompromised and immunocompetent patients. Epizootics and genetic diversity of USUV in different bird species as well as detection of the virus in mosquitoes suggest repeated USUV introductions into Europe with endemization in some countries. The zoonotic potential of USUV has been reported in a growing number of human cases. Clinical cases of neuroinvasive disease and USUV fever, as well as seroconversion in blood donors were reported in Europe since 2009. While most USUV strains detected in humans, birds and mosquitoes belong to European USUV lineages, several reports indicate the presence of African lineages as well. Since spreading trends of USUV are likely to continue, continuous multidisciplinary interventions (“One Health” concept) should be conducted for monitoring and prevention of this emerging arboviral infection.

Introduction: With the shift of research focus to personalized medicine in Alzheimer's Dementia (AD), there is an urgent need for tools that are capable of quantifying a patient's risk using diagnostic biomarkers. The Medical Informatics Platform (MIP) is a distributed e-infrastructure federating large amounts of data coupled with machine-learning (ML) algorithms and statistical models to define the biological signature of the disease. The present study assessed (i) the accuracy of two ML algorithms, i.e., supervised Gradient Boosting (GB) and semi-unsupervised 3C strategy (Categorize, Cluster, Classify-CCC) implemented in the MIP and (ii) their contribution over the standard diagnostic workup. Methods: We examined individuals coming from the MIP installed across 3 Italian memory clinics, including subjects with Normal Cognition (CN, n = 432), Mild Cognitive Impairment (MCI, n = 456), and AD (n = 451). The GB classifier was applied to best discriminate the three diagnostic classes in 1,339 subjects, and the CCC strategy was used to refine the classical disease categories. Four dementia experts provided their diagnostic confidence (DC) of MCI conversion on an independent cohort of 38 patients. DC was based on clinical, neuropsychological, CSF, and structural MRI information and again with addition of the outcome from the MIP tools. Results: The GB algorithm provided a classification accuracy of 85% in a nested 10-fold cross-validation for CN vs. MCI vs. AD discrimination. Accuracy increased to 95% in the holdout validation, with the omission of each Italian clinical cohort out in turn. CCC identified five homogeneous clusters of subjects and 36 biomarkers that represented the disease fingerprint. In the DC assessment, CCC defined six clusters in the MCI population used to train the algorithm and 29 biomarkers to improve patients staging. GB and CCC showed a significant impact, evaluated as +5.99% of increment on physicians' DC. The influence of MIP on DC was rated from "slight" to "significant" in 80% of the cases. Discussion: GB provided fair results in classification of CN, MCI, and AD. CCC identified homogeneous and promising classes of subjects via its semi-unsupervised approach. We measured the effect of the MIP on the physician's DC. Our results pave the way for the establishment of a new paradigm for ML discrimination of patients who will or will not convert to AD, a clinical priority for neurology.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. Bluetongue virus serotype 8 (BTV-8), an arthropod-borne virus of ruminants, emerged in livestock in northern Europe in 2006, spreading to most European countries by 2009 and causing losses of billions of euros. Although the outbreak was successfully controlled through vaccination by early 2010, puzzlingly, a closely related BTV-8 strain re-emerged in France in 2015, triggering a second outbreak that is still ongoing. The origin of this virus and the mechanisms underlying its re-emergence are unknown. Here, we performed phylogenetic analyses of 164 whole BTV-8 genomes sampled throughout the two outbreaks. We demonstrate consistent clock-like virus evolution during both epizootics but found negligible evolutionary change between them. We estimate that the ancestor of the second outbreak dates from the height of the first outbreak in 2008. This implies that the virus had not been replicating for multiple years prior to its re-emergence in 2015. Given the absence of any known natural mechanism that could explain BTV-8 persistence over this long period without replication, we hypothesise that the second outbreak could have been initiated by accidental exposure of livestock to frozen material contaminated with virus from approximately 2008. Our work highlights new targets for pathogen surveillance programmes in livestock and illustrates the power of genomic epidemiology to identify pathways of infectious disease emergence., SCOPUS: ar.j, info:eu-repo/semantics/published

The epidemiological patterns of Bluetongue (BT) in North Africa and Mediterranean Basin (MB) dramatically changed by emergence of subsequent episodes of novel bluetongue virus (BTV) serotypes with highly pathogenic indexes and socio‐economic impacts. The objective of the study was to investigate the sero‐prevalence and serotype distribution of BTV in Libya. During 2015‐2016, a total of 826 serum samples were collected from domestic ruminants in Libya. All sera were assayed by competitive enzyme‐linked immunosorbent assays (c‐ELISA). C‐Elisa‐positive samples (43.3%; 173/400) were further analyzed by virus neutralization assay to identify BTV serotypes and determine the antibody titre of positive samples. An overall BTV sero‐prevalence was 48.4% (95% CI: 45.0%‐51.8%). Neutralizing antibodies were detected against the following BTV serotypes namely: BTV‐1, BTV‐2, BTV‐3, BTV‐4, BTV‐9 and BTV‐26. While BTV‐1, BTV‐2, BTV‐4 and BTV‐9 circulation was unsurprising as they have been responsible of the last year outbreaks in Northern African Countries, the detection of BTV‐3 and BTV‐26 was definitely new and concerning for the animal health of the countries facing the Mediterranean Basin. It is crucial that European and Northern African authorities collaborate in organizing common surveillance programmes to early detect novel strains or emerging serotypes in order to set up proper preventive measures, and, in case, develop specific vaccines and plan coordinated vaccination campaigns.

The aim of this study was to develop a real-time RT-PCR to detect and quantitate feline morbillivirus (FeMV) RNA in biological samples. Primers and probe were targeted on a conserved region of FeMV P/V/C gene. To validate the assay with field samples, a total number of specimens of cats have been recruited including 264 urine and blood samples and compared with a generic RT-PCR targeting the L protein encoding gene of morbilliviruses. In addition, 385 tissue samples from 35 carcasses of cats have been also employed. RNA titres were low in all tested samples. Results also indicated the absence of cross-reaction with related morbilliviruses and existing pathogens of cats. In tissues with low levels of FeMV RNA, the presence of viral antigen was also evidenced by immunohistochemistry targeting the N viral protein. This newly described assay allows for a rapid, accurate and reliable quantitative detection of FeMV RNA that can be applied for diagnostics and research studies.

Bluetongue (BT), caused by Bluetongue virus (BTV), is a disease that affects ruminants such as cattle, sheep, goats and deer. BTV is transmitted by female midges of the genus Culicoides. In Brazil, information on the prevalence of BTV in cattle is limited, so the objective of this work was to identify BTV serotypes in cattle. The State of São Paulo was divided into seven cattle‐producing regions, and in each of them, 300 cattle farms were randomly selected. One animal from each farm (out of a total of 1,598 farms) was selected and its sera tested by virus neutralization technique against BTV serotypes (1–24 and 26) for determining antibody titre. Moreover, for each sampled farm, an epidemiological questionnaire was submitted to verify the type of cattle production and the zootechnical and sanitary practices carried out, which could be associated with a higher risk of BTV infection. In this study, antibodies (percentage, [95% confidence interval]) were identified against 11 serotypes: BTV‐1 (22.15%, [15.72–27.92]), BTV‐2 (31.03%, [26.65–37.98]), BTV‐3 (18.96%, [12.42–24.90]), BTV‐4 (24.90% [19.41–29.12]), BTV‐9 (6.82%, [1.45–11.72]), BTV‐12 (7.50%, [2.82–12.51]), BTV‐17 (23.90%, [17.35–29.35]), BTV‐19 (10.20%, [4.62–5.56]), BTV‐21 (30.66%, [25.00–36.00]), BTV‐22 (12.14%, [5.91–18.55]), BTV‐26 (57.00%, [51.41–63.59]). In this study, for the first time in Brazil serological evidence of the presence of serotypes BTV‐2, BTV‐9, BTV‐21 and BTV‐26 is reported. The variable ‘new cattle entering herd’ was considered a risk factor for the occurrence of infection (OR = 2.183, 95% CI = 1.6–2.9).

Mosquito-borne diseases (MBDs) are spreading worldwide due to globalization and climate change, representing a threat for both humans and animals. Of great concern are the infections caused by viruses belonging to the Flavivirus genus as West Nile virus (WNV) and Usutu virus (USUV) transmitted by Culex sp. or Dengue virus and Zika virus (ZIKV), transmitted by Aedes sp. This work describes the surveillance protocol enforced in Piedmont (Northwestern Italy) to control MBDs spread, focusing on the activities performed on mosquitoes during the 2015 vector season.From July to October, mosquitoes were fortnightly sampled in 50 selected sites according to risk factors with CDC dry ice-baited traps and BG-Sentinel traps baited with BG-Lure and dry ice. Adults were counted, identified to species level, pooled, and screened for flaviviruses using different reverse transcription-PCR protocols and sequencing. Finally, phylogenetic analysis was performed on a dataset including 2014 and 2015 WNV sequences and reference sequences retrieved from GenBank.A total of 17,000 mosquitoes, grouped in 730 pools, were tested. Five pools of Culex pipiens were positive for WNV Lineage 2 in Novara, Alessandria, Vercelli, and Torino Provinces. One pool of C. pipiens and one pool of Anopheles maculipennis s.l. were positive for USUV in Vercelli and Alessandria Provinces. In Vercelli Province one pool of C. pipiens resulted positive both for WNV and USUV. Control measures were quickly implemented. Phylogenetic analyses showed that the WNV Lin 2 sequences from Piedmont region cluster with those circulating in Northeastern Italy in the previous years. Given the positive trend in WNV activity compared to 2014 and the emergence caused by other flavivirus as ZIKV, the level of attention for the 2016 vector season may be increased and this surveillance protocol could represent an important tool for public health authorities.

To monitor the spread and to evaluate the role for public health of Usutu virus (USUV) in an endemic area of Italy.The survey was retrospectively conducted by detecting USUV RNA and USUV antibodies in cerebrospinal fluid and serum samples collected between 2008 and 2011 from 915 patients with or without neurologic impairments in the area of the municipality of Modena, Italy. Organs of birds and pools of mosquitoes were also tested for USUV RNA. Positive samples were partially sequenced and used for phylogenetic analysis.The presence of USUV RNA (1.1%; 95% confidence interval (CI) 0.6-2.0) was significantly (p 0.05) higher than that of West Nile virus (0%; 95% CI 0-0.33). USUV antibody level was 6.57% (95% CI 4.87-8.82), and it was significantly higher (p0.05) compared to that of West Nile virus (p 2.96, 95% CI 1.89-4.62). Partial genome sequencing of USUV strains detected in humans, birds and mosquitoes revealed high nucleotide sequence identity within them and with the USUV strains isolated in Central Europe.USUV infection in humans is not a sporadic event in the studied area, and USUV neuroinvasiveness has been confirmed.

A collection of 3069 human sera collected in the area of the municipality of Modena, Emilia Romagna, Italy, was retrospectively investigated for specific antibodies against Usutu (USUV) and West Nile viruses (WNV). All the samples resulting positive using a preliminary screening test were analyzed with the plaque reduction neutralization test. Overall, 24 sera were confirmed as positive for USUV (0.78%) and 13 for WNV (0.42%). The results suggest that in 2012, USUV was circulating more than WNV in North-eastern Italy.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. Bluetongue virus serotype 8 (BTV-8), an insect-borne virus of ruminants, emerged in livestock in Northern Europe in 2006, spreading to most European countries by 2009 and causing losses of billions of Euros. Though the outbreak was successfully controlled through vaccination by early 2010, puzzlingly a closely-related BTV-8 strain re-emerged in France in 2015, triggering a second outbreak that is still ongoing. The origin of this virus and the mechanisms underlying its re-emergence are unknown. Here, we performed phylogenetic analyses of 164 whole BTV-8 genomes sampled throughout the two outbreaks. We demonstrate consistent clock-like virus evolution during both epizootics but found negligible evolutionary change between them. We estimate that the ancestor of the second outbreak dates from the height of the first outbreak in 2008. This implies that the virus had not been replicating for multiple years prior to its re-emergence in 2015. Given the absence of any known natural mechanism that could explain BTV-8 persistence over this period without replication, we conclude that the second outbreak was most likely initiated by accidental exposure of livestock to frozen material contaminated with virus from approximately 2008. Our work highlights new targets for pathogen surveillance programmes in livestock and illustrates the power of genomic epidemiology to identify pathways of infectious disease emergence.

The epidemiology of West Nile (WNV) and Usutu virus (USUV) has changed dramatically over the past two decades. Since 1999, there have been regular reports of WNV outbreaks and the virus has expanded its area of circulation in many Southern European countries. After emerging in Italy in 1996, USUV has spread to other countries causing mortality in several bird species. In 2009, USUV seroconversion in horses was reported in Italy. Co-circulation of both viruses was detected in humans, horses and birds. The main vector of WNV and USUV in Europe is Culex pipiens, however, both viruses were found in native Culex mosquito species (Cx. modestus, Cx. perexiguus). Experimental competence to transmit the WNV was also proven for native and invasive mosquitoes of Aedes and Culex genera (Ae. albopictus, Ae. detritus, Cx. torrentium). Recently, Ae. albopictus and Ae. japonicus naturally-infected with USUV were reported. While neuroinvasive human WNV infections are well-documented, USUV infections are sporadically detected. However, there is increasing evidence of a role of USUV in human disease. Seroepidemiological studies showed that USUV circulation is more common than WNV in some endemic regions. Recent data showed that WNV strains detected in humans, horses, birds, and mosquitoes mainly belong to lineage 2. In addition to European USUV lineages, some reports indicate the presence of African USUV lineages as well. The trends in WNV/USUV range and vector expansion are likely to continue in future years. This mini- review provides an update on the epidemiology of WNV and USUV infections in Southern Europe within a multidisciplinary "One Health" context.

Here we report studies of the antigenic relationship of West Nile virus (WNV) and Usutu virus (USUV), two zoonotic flaviviruses from Italy, together with a Japanese encephalitis virus (JEV) strain and compared them with their genetic relationship using the immunodominant viral E protein. Thirty-nine isolates and reference strains were inactivated and used to immunize rabbits to produce hyper immune sera. Serum samples were tested by neutralization against all isolates and results visualized by generating antigenic map. Strains of WNV, USUV, and JEV grouped in separate clusters on the antigenic map. JEV was closer antigenically to USUV (mean of 3.5 Antigenic Unit, AU, equivalent to a 2-fold change in antibody titer) than to WNV strains (mean of 6 AU). A linear regression model predicted, on average, one unit of antigenic change, equivalent to a 2-fold change in antibody titer, for every 22 amino acid substitutions in the E protein ectodomain. Overall, antigenic map was demonstrated to be robust and consistent with phylogeny of the E protein. Indeed, the map provided a reliable means of visualizing and quantifying the relationship between these flaviviruses. Further antigenic analyses employing representative strains of extant serocomplexes are currently underway. This will provide a more in deep knowledge of antigenic relationships between flaviviruses.

Over the last 20 years, Italy has experienced multiple incursions of different serotypes of Bluetongue virus (BTV), a Culicoides‐borne arbovirus, the causative agent of bluetongue (BT), a major disease of ruminants. The majority of these incursions originated from Northern Africa, likely because of wind‐blown dissemination of infected midges. Here, we report the first identification of BTV‐3 in Sardinia, Italy. BTV‐3 circulation was evidenced in sentinel animals located in the province of Sud Sardegna on September 19, 2018. Prototype strain BTV‐3 SAR2018 was isolated on cell culture. BTV‐3 SAR2018 sequence and partial sequences obtained by next‐generation sequencing from nucleic acids purified from the isolate and blood samples, respectively, were demonstrated to be almost identical (99–100% of nucleotide identity) to BTV‐3 TUN2016 identified in Tunisia in 2016 and 2017, a scenario already observed in past incursions of other BTV serotypes originating from Northern Africa.

Data on the immune response to West Nile virus (WNV) are limited. We analyzed the antiviral cytokine response in serum and cerebrospinal fluid (CSF) samples of patients with WNV fever and WNV neuroinvasive disease using a multiplex bead-based assay for the simultaneous quantification of 13 human cytokines. The panel included cytokines associated with innate and early pro-inflammatory immune responses (TNF-α/IL-6), Th1 (IL-2/IFN-γ), Th2 (IL-4/IL-5/IL-9/IL-13), Th17 immune response (IL-17A/IL-17F/IL-21/IL-22) and the key anti-inflammatory cytokine IL-10. Elevated levels of IFN-γ were detected in 71.7% of CSF and 22.7% of serum samples (p = 0.003). Expression of IL-2/IL-4/TNF-α and Th1 17 cytokines (IL-17A/IL-17F/IL-21) was detected in the serum but not in the CSF (except one positive CSF sample for IL-17F/IL-4). While IL-6 levels were markedly higher in the CSF compared to serum (CSF median 2036.71, IQR 213.82–6190.50, serum median 24.48, IQR 11.93–49.81, p &lt, 0.001), no difference in the IL-13/IL-9/IL-10/IFN-γ/IL-22 levels in serum/CSF was found. In conclusion, increased concentrations of the key cytokines associated with innate and early acute phase responses (IL-6) and Th1 type immune responses (IFN-γ) were found in the CNS of patients with WNV infection. In contrast, expression of the key T-cell growth factor IL-2, Th17 cytokines, a Th2 cytokine IL-4 and the proinflammatory cytokine TNF-α appear to be concentrated mainly in the periphery.

Circoviruses are relatively novel pathogens with increased importance in canids. In this study, we first screened the presence of dog circovirus (DogCV) by molecular methods from a total number of 389 internal organ samples originating from 277 individuals of domestic dogs and wild animals including wolves, foxes and badgers. All the animals originated from Central-Southern Italy, specifically from Abruzzi and Molise regions, areas hosting several natural parks. DogCV was detected in 9/34 wolves (P=26.4%; IC 95%: 14.6–43.1%), 8/209 dogs (P=3.8%; IC 95%: 1.9–7.3%), 0/24 foxes (P=0%; IC 95%: 0–13.8%), 1/10 badgers (P=10%; IC 95%: 1.79–40.4%). However, all DogCV positive animals were shown to be infected at least by an additional key pathogen, including canine distemper virus (CDV) and canine parvovirus type 2. All wolves, but one, presenting DogCV in the internal tissues suffered from CDV infection. The DNA purified from 17 DogCV infected organs was used for whole genome sequencing and phylogenetic analysis.

By late March 2020, Villa Caldari, a small village of the municipality of Ortona (Abruzzo region), was registering an incidence rate of COVID-19 cases ten times greater than the overall municipality and was declared a hotspot area. Twenty-two days later, epidemiological investigation and sampling were performed, to evaluate SARS-CoV-2 circulation and the presence of SARS-CoV-2 antibodies. Overall, 681 nasopharyngeal swabs and 667 blood samples were collected. Only one resident of the village resulted in being positive for RNA viral shedding, while 73 were positive for SARS-CoV-2 antibodies. The overall seroprevalence was 10.9%. The difference between the seroprevalence of infection in asymptomatic and symptomatic individuals was significant (&chi, 2 = 14.50 p-value = 0.0001). Amongst the residents positive for antibodies, fatigue and/or muscle pain, fever and anosmia were the most experienced symptoms, whose most frequent onset was observed during the first two weeks of March. Familial and habit-related clusters were highlighted. Nevertheless, the investigations showed a low SARS-CoV-2 circulation in the village at the time of the sampling, demonstrating virus transmission could be limited when strict emergency measures are followed. Given the favorable results, the emergency measures were then lifted.

The continuous flow of billions of birds between Africa and Europe creates an "ecological bridge" between physically remote areas. Migratory birds fly south from their breeding grounds during late summer/fall and fly back in spring. These movements regulate the spread of internal and external parasites, as well as pathogens of potential public health concern. The aim of the present study was to investigate the possible introduction of exotic tick species and tick-borne pathogens into Europe via migratory birds. At the bird observatory of Ventotene island (Italy), 443 feeding ticks were collected from 249 birds captured and ringed during their northbound migration in spring 2013. Each tick was identified by morphological and molecular methods and then tested for bacterial and viral pathogens: Borrelia burgdorferi s.l., Rickettsia spp., Ehrlichia ruminantium and Coxiella burnetii, Crimean Congo haemorrhagic fever virus (CCHFV) and Flavivirus. Morphological and molecular identification confirmed Hyalomma rufipes as the most abundant species among the collected arthropods (366/443; 82.6%) followed by Hyalomma marginatum (10/433; 2.3%). Rickettsia aeschlimannii was identified in 158 ticks, while one engorged Amblyomma variegatum nymph was infected with Rickettsia africae. The other bacteria were not detected in any specimen. Among viruses, RNA belonging to West Nile virus and other Flavivirus were detected whereas all ticks were negative for CCHFV RNA. These results confirm how migratory birds play a role in carrying Rickettsia-infected ticks, as well as viruses of zoonotic importance, from Africa into Europe. To what extent tick species are capable of establishing a permanent population once introduced in naive areas, is far from defined and deserve further investigation.