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African swine fever (ASF), characterized by high mortality rates in infected animals, remains a significant global veterinary and economic concern, due to the widespread distribution of ASF virus (ASFV) genotype II across five continents. In this study, ASFV strains collected in Italy during 2022-2023 from two geographical clusters, North-West (Alessandria) and Calabria, were fully sequenced. In addition, an in vivo experiment in pigs was performed. Complete genomic sequencing of 30 strains revealed large-fragment deletions and translocations. In Alessandria, five samples showed two different deletions in the 5' genomic region: a ~4340 bp deletion (positions ~9020-13,356 in Georgia 2007/1) in four samples and a 2162 bp deletion (positions 17,837-19,998) in one sample. Another strain showed a truncation of 1950 bp at the 3' end. In Calabria, strains showed a 5137 bp deletion (positions 10,755-15,891) and ~2 kb truncations in the 3' region. Two strains showed a translocation from the region 1-2244 to positions 188,631-190,584. In vivo characterization of the deleted strain 22489.4_2312/RC/2023 revealed identical disease progression to the wild-type strain, with severe ASF symptoms in inoculated pigs. This study is the first to report natural deleted strains of ASFV in Italy, revealing unique genomic deletions distinct from those in previously known strains.

Biting midges ( Culicoides spp.) are important vectors of several insect borne arboviruses but are underrepresented in terms of availability of high-resolution genomic resources. We assembled and annotated complete mitochondrial genomes for two Culicoides species, namely C. brevitarsis and C. imicola which are proven vectors for Bluetongue Virus (BTV). We used both long read and short read sequencing technologies to assemble the circular genomes. The genome sizes are 17,100 bp and 17,031 bp, respectively, all comprising 37 genes, including 13 protein, 22 tRNA, two rRNA coding genes, and one non-coding AT rich control region. The gene organizations and orientations are comparable to other available Culicoides mitogenomes, except for a translocation of ND2 and six tRNA genes in both C. brevitarsis and C. imicola . Eleven protein-coding genes encode a full TAA stop codon, while two ( ND5 , COX3 ) are completed by mRNA polyadenylation. Phylogenetic analysis of the mitogenomes showed C. brevitarsis and C. imicola form a monophyletic group. The sequences of these mitogenomes contribute to a baseline of molecular tools for diagnostics and surveillance for use by World Organisation for Animal Health (WOAH) reference laboratories for monitoring vectors of emerging diseases., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2025 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

West Nile virus (WNV) is a mosquito-borne pathogen that can infect humans, equids, and many bird species, posing a threat to their health. It consists of eight lineages, with Lineage 1 (L1) and Lineage 2 (L2) being the most prevalent and pathogenic. Italy is one of the hardest-hit European nations, with 330 neurological cases and 37 fatalities in humans in the 2021-2022 season, in which the L1 re-emerged after several years of low circulation. We assembled a database comprising all publicly available WNV genomes, along with 31 new Italian strains of WNV L1 sequenced in this study, to trace their evolutionary history using phylodynamics and phylogeography. Our analysis suggests that WNV L1 may have initially entered Italy from Northern Africa around 1985 and indicates a connection between European and Western Mediterranean countries, with two distinct strains circulating within Italy. Furthermore, we identified new genetic mutations that are typical of the Italian strains and that can be tested in future studies to assess their pathogenicity. Our research clarifies the dynamics of WNV L1 in Italy, provides a comprehensive dataset of genome sequences for future reference, and underscores the critical need for continuous and coordinated surveillance efforts between Europe and Africa.

Coxiella burnetii is the causative agent of Q fever. The main reservoirs for this bacterium, which can lead to human infection, in our region are typically cattle, goats, and sheep. In animals, C. burnetii infection is often detected due to reproductive problems. European Member States are required to report confirmed cases annually, but the lack of uniform reporting methods makes the data rather inconsistent. The Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise is involved in official controls to identify the causes of abortions, monitor suspected or positive herds, evaluate suspected infections in pets and humans, monitor the spread in wildlife, etc. In this paper, we summarize the presence of C. burnetii over the last five years (2019-2023). Additionally, a detailed overview of C. burnetii infection in wild and domestic animals is provided. Five hundred sixty animals-including cattle; goats; sheep; wild animals, such as deer, boars, wolves, roe deer, owls, and otters; buffalo; dogs; horses; cats; and a donkey-and six human samples were tested by real-time PCR on the transposase gene IS1111 to detect C. burnetii . The MST profile was identified in some of the samples. Outbreaks of C. burnetii occurred in four herds. In one of them, it was possible to follow the outbreak from inception to eradication by evaluating the effect of vaccination on real-time PCR Ct values. A total of 116 animals tested positive for C. burnetii , including 73 goats, 42 sheep, and one bovine. None of the other samples tested positive. The strains for which the ST was performed were identified as ST79, a strain that has been present in the area for more than ten years. The effect of vaccination on the reduction of positive samples and the variation of real-time PCR Ct values was evaluated in strict correlation.

Usutu virus (USUV), an arbovirus from the Flaviviridae family, genus Flavivirus, has recently gained increasing attention because of its potential for emergence. After his discovery in South Africa, USUV spread to other African countries, then emerged in Europe where it was responsible for epizootics. The virus has recently been found in Asia. USUV infection in humans is considered to be most often asymptomatic or to cause mild clinical signs. However, a few cases of neurological complications such as encephalitis or meningo-encephalitis have been reported in both immunocompromised and immunocompetent patients. USUV natural life cycle involves Culex mosquitoes as its main vector, and multiple bird species as natural viral reservoirs or amplifying hosts, humans and horses can be incidental hosts. Phylogenetic studies carried out showed eight lineages, showing an increasing genetic diversity for USUV. This work describes the development and validation of a novel whole-genome amplicon-based sequencing approach to Usutu virus. This study was carried out on different strains from Senegal and Italy. The new approach showed good coverage using samples derived from several vertebrate hosts and may be valuable for Usutu virus genomic surveillance to better understand the dynamics of evolution and transmission of the virus., (© 2024. The Author(s).)

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.

The spread of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is a major public health issue. Bivalves are filter-feeder animals capable of bioaccumulating the microorganisms present in water. This physiological characteristic makes them both good indicators of environmental contamination and possible carriers of pathogenic bacteria, including those resistant to antimicrobials. The aim of this study was to investigate the occurrence of ESBL-producing E. coli in clams ( n  = 308) collected from harvesting areas of the Central Adriatic Sea between 2018 and 2019. ESBL- /class C β-lactamase (AmpC)- producing E. coli and Escherichia spp. were isolated by streaking over the surface of MacConkey agar plates supplemented with cefotaxime enriched broths of the initial shellfish suspension. E. coli and Escherichia spp. resistant to cefotaxime were screened for ESBL production by using the double disk synergy test. Susceptibility to different antimicrobials and confirmation of ESBL-production were determined by the minimum inhibitory concentration (MIC) test. Isolates were further characterized by whole genome sequencing (WGS) and bioinformatic analysis of genomes with different tools. Overall, ESBL-producing E. coli were isolated from 3% of the samples. Of 13 ESBL- and ESBL-/AmpC-producing Escherichia spp. ( n  = 11 E. coli , n  = 1 E. marmotae , n  = 1 E . ruysiae ) isolates, 13 were resistant to ampicillin and cefotaxime, 9 to sulfamethoxazole, 6 to tetracycline and nalidixic acid, 4 to trimethoprim, and 3 to ceftazidime, cefoxitin, ciprofloxacin, and chloramphenicol. Moreover, the majority (8/11) of the ESBL-producing E. coli isolates were multidrug-resistant. WGS showed that the isolates predominantly carried the bla CTX-M-15 gene (3/11) and bla CTX-M-14 and bla CTX-M-1 (2/11 each). The AmpC β-lactamase CMY-2 was found in two isolates. Phylogroup A was the most prevalent (5/11), followed by phylogroups D (4/11), F (1/11), and B2 (1/11). Ten different sequence types (STs) were identified. Occurrence at sampling sites ranged between 0 and 27%. To identify associations between the occurrence of ESBL-producing E. coli and E. coli levels, samples were divided into two groups, with E. coli at >230 MPN/100 g and E. coli at ≤230 MPN/100 g. ESBL-producing E. coli isolates were significantly more commonly recovered in samples with higher E. coli levels (14%) than in those with lower levels of E. coli (2%). Moreover, the majority (3/4) of the potentially pathogenic strains were isolated in samples with higher E. coli levels. These findings provided evidence for the bacterial indicator of fecal contamination, E. coli , as an index organism for ESBL-producing E. coli isolates in bivalves., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Leoni, Sacchini, Pieralisi, Angelico, Magistrali, Cucco, Massacci, Albini, Duranti, Cammà, Secondini, Rinaldi and Barchiesi.)

Within public health control strategies for SARS-CoV-2, whole genome sequencing (WGS) is essential for tracking viral spread and monitoring the emergence of variants which may impair the effectiveness of vaccines, diagnostic methods, and therapeutics. In this manuscript different strategies for SARS-CoV-2 WGS including metagenomic shotgun (SG), library enrichment by myBaits® Expert Virus-SARS-CoV-2 (Arbor Biosciences), nCoV-2019 sequencing protocol, ampliseq approach by Swift Amplicon® SARS-CoV-2 Panel kit (Swift Biosciences), and Illumina COVIDSeq Test (Illumina Inc.), were evaluated in order to identify the best approach in terms of results, labour, and costs. The analysis revealed that Illumina COVIDSeq Test (Illumina Inc.) is the best choice for a cost-effective, time-consuming production of consensus sequences., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

It is unclear whether West Nile virus (WNV) circulates between Africa and Europe, despite numerous studies supporting an African origin and high transmission in Europe. We integrated genomic data with geographic observations and phylogenetic and phylogeographic inferences to uncover the spatial and temporal viral dynamics of WNV between these two continents. We focused our analysis towards WNV lineages 1 (L1) and 2 (L2), the most spatially widespread and pathogenic WNV lineages. Our study shows a Northern-Western African origin of L1, with back-and-forth exchanges between West Africa and Southern-Western Europe; and a Southern African origin of L2, with one main introduction from South Africa to Europe, and no back introductions observed. We also noticed a potential overlap between L1 and L2 Eastern and Western phylogeography and two Afro-Palearctic bird migratory flyways. Future studies linking avian and mosquito species susceptibility, migratory connectivity patterns, and phylogeographic inference are suggested to elucidate the dynamics of emerging viruses., (© 2023. Springer Nature Limited.)

West Nile virus is a re-emerging arbovirus whose impact on public health is increasingly important as more and more epidemics and epizootics occur, particularly in America and Europe, with evidence of active circulation in Africa. Because birds constitute the main reservoirs, migratory movements allow the diffusion of various lineages in the world. It is therefore crucial to properly control the dispersion of these lineages, especially because some have a greater health impact on public health than others. This work describes the development and validation of a novel whole-genome amplicon-based sequencing approach to West Nile virus. This study was carried out on different strains from lineage 1 and 2 from Senegal and Italy. The presented protocol/approach showed good coverage using samples derived from several vertebrate hosts and may be valuable for West Nile genomic surveillance.

In January 2022, West Nile virus (WNV) lineage 2 (L2) was detected in an adult female goshawk rescued near Perugia in the region of Umbria (Italy). The animal showed neurological symptoms and died 15 days after its recovery in a wildlife rescue center. This was the second case of WNV infection recorded in birds in the Umbria region during the cold season, when mosquitoes, the main WNV vectors, are usually not active. According to the National Surveillance Plan, the Umbria region is included amongst the WNV low-risk areas. The necropsy evidenced generalized pallor of the mucous membranes, mild splenomegaly, and cerebral edema. WNV L2 was detected in the brain, heart, kidney, and spleen homogenate using specific RT-PCR. Subsequently, the extracted viral RNA was sequenced. A Bayesian phylogenetic analysis performed through a maximum-likelihood tree showed that the genome sequence clustered with the Italian strains within the European WNV strains among the central-southern European WNV L2 clade. These results, on the one hand, confirmed that the WNV L2 strains circulating in Italy are genetically stable and, on the other hand, evidenced a continuous WNV circulation in Italy throughout the year. In this report case, a bird-to-bird WNV transmission was suggested to support the virus overwintering. The potential transmission through the oral route in a predatory bird may explain the relatively rapid spread of WNV, as well as other flaviviruses characterized by similar transmission patterns. However, rodent-to-bird transmission or mosquito-to-bird transmission cannot be excluded, and further research is needed to better understand WNV transmission routes during the winter season in Italy.

Since the very beginning of the COVID-19 pandemic, SARS-CoV-2 detection has been described in several animal species. A total of 625 outbreaks in animals have been reported globally, affecting 17 species in 32 countries and the human source of infection has been recognized including pet owners, zookeepers, and farmers. In this report, we describe the case of a paucisymptomatic dog in Italy infected with SARS-CoV-2 from a household with three confirmed human cases of COVID-19 living in Pesaro (Marche region, Italy). The dog showed high viral RNA titers in the nasal and oropharyngeal swabs. In the nasal swab, SARS-CoV-2 RNA lasted for a least a week. By sequencing, the strain was assigned to the AY.23 lineage (PANGO), one of the sub-lineages of the major SARS-CoV-2 Delta variant of concern (VOC). Although we did not process the swabs of the three human cases, we strongly suspect a human origin for the dog infection. In this regard, AY.23 sequences, although never released thus far in the Marche region, were detected in the neighboring regions. Our findings highlight once more the need for a One Health approach for SARS-CoV-2 surveillance, management, and control, thus preventing viral spillover from animals to humans.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. We report the complete sequences of three SARS-CoV-2 P.1 strains obtained from nasopharyngeal swab specimens from three patients returning from Brazil to Italy., (Copyright © 2021 Delli Compagni et al.)