An amplicon-based sequencing approach for Usutu virus characterization.

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.

Disseminated Scedosporium apiospermum infection in a Maremmano-Abruzzese sheepdog.

BACKGROUND: Few cases of scedosporiosis have been reported in animals, but the true prevalence is probably underestimated due to a lack of awareness. Scedosporiosis in dogs has often been associated with localized infection (i.e., nasal infection, eumycetoma, or keratomycosis) or, in rare cases, disseminated infections. CASE PRESENTATION: This case report describes the clinical and pathological features and the diagnostic process of a rare systemic and fatal fungal infection in a dog caused by Scedosporium apiospermum. A 10-month-old female Maremmano-Abruzzese sheepdog showing weakness, lethargy, lateral decubitus, miosis and muscular rigidity was presented. Rodenticide poisoning was clinically suspected for the differential diagnosis. However, postmortem examinations revealed the presence of a swollen and soft subcutaneous nodule located near the right inguinal breast, which was associated with massive enlargement of the inguinal lymph nodes and small disseminated, cream-colored nodules in the kidneys and mesentery. Multiple fungal pyogranulomas were observed upon histological examination. Fungal isolation from the kidneys, breast and inguinal lymph nodes was performed. The internal transcribed spacer (ITS) sequences from the fungal colony DNA were searched in BLAST in the NCBI GenBank for species identification. The sequences of the fungi isolated from the kidney and breast cultures showed 100% sequence identity with sequences from Scedosporium apiospermum. CONCLUSIONS: This report shows that Scedosporium apiospermum may act as a primary pathogen in young and apparently healthy dogs and represents an important pathogen that should be considered during the diagnostic process, particularly when a fungal infection is suspected.

Aphanomyces astaci genotypes involved in recent crayfish plague outbreaks in central Italy.

The oomycete Aphanomyces astaci is the causative agent of crayfish plague in native European freshwater crayfish. Molecular analyses showed that several distinct genotype groups of this pathogen, apparently associated with different original host taxa, are present in Europe. Tracking their distribution may contribute to understanding the introduction pathways of A. astaci. We used microsatellite markers to genotype the pathogen strains involved in 7 mass mortalities of the endangered indigenous crayfish Austropotamobius pallipes that occurred between 2009 and 2016 in the Abruzzi and Molise regions, central Italy. Three A. astaci genotype groups (A, B, and D, with the latter represented by 2 distinct multilocus genotypes) were identified, suggesting the existence of multiple infection sources even in a relatively small area. Most crayfish plague episodes were due to genotype groups associated with the North American host species Pacifastacus leniusculus and Procambarus clarkii, although these crayfish are not widespread in the study area. A. astaci genotype group A was detected not only in crayfish plague outbreaks but also in apparently healthy Astacus leptodactylus imported for human consumption from Armenia and kept alive in an aquaculture facility. Imports of chronically infected A. leptodactylus from Armenia, Turkey, and possibly Eastern Europe are an underestimated introduction pathway for A. astaci. Although we cannot exclude the presence of latently infected native populations of A. pallipes in the region, A. astaci infections in legally imported crayfish species considered vulnerable to crayfish plague may represent further reservoirs of A. astaci; this should be reflected in the policies regulating the trade of live crayfish.

Detection of vesivirus in minks (Neovison vison), Italy 2021.

Vesiviruses are important animal pathogens with a broad host range, and they have also been involved in accidental contamination of cells used for the production of drugs for rare and life-threatening human diseases. A vesivirus (family Caliciviridae) was detected in minks (Neovison vison) with respiratory and neurological signs, during syndromic surveillance for SARS-CoV-2 conducted in Italy. The complete genome (8,397 nucleotides in length) of the vesivirus strain ITA/2021/mink/TE (OR130287) was obtained by combining NGS approach with 5' and 3' RACE protocols. The virus was seemingly more related (95.9-97.2% nt identity in the partial RNA-dependent RNA polymerase) to American vesivirus isolates 9/1980/US, 12/1980/US, and 20/1980/US dating back to the early 1980s than to recent mink strains. These results highlight the importance of gathering information on the virome of animals.

Low pathogenic human coronaviruses during the first waves of COVID-19 in Italy.

Low-pathogenic human coronaviruses (HCoVs) infect the upper respiratory tract and cause mild, cold-like respiratory illness. Although several studies have shown evidence of the global distribution of HCoVs, information about their distribution in Italy are often focused only on hospitalized children and elderly with respiratory symptoms. In this study, a total of 916 swab samples collected during the first two SARS-CoV-2 pandemic waves in Abruzzo region (central Italy) was selected for molecular screening of low pathogenic HCoVs by real-time RT-PCR. We identified low-pathogenic HCoV in nine samples. Positive samples underwent whole genome sequencing for genome characterization; indeed, we also report the whole genome sequence of a HCoV-229E strain.

Genomic Characterization of a Wild-Type Bovine alphaherpesvirus 1 (BoAHV-1) Strain Isolated in an Outbreak in Central Italy.

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.

Identification and characterization of two atypical strains of bluetongue virus in sheep, Tunisia.

Bluetongue virus (BTV) is the causative agent of the bluetongue disease (BT), an infectious disease of domestic and wild ruminants that is primarily transmitted by Culicoides biting midges. In recent years, several novel BTV serotypes (often referred to as "atypical" BTVs) have been documented. These strains are usually asymptomatic in animals and seem to be unable to replicate efficiently in the arthropod vector. Here we report the detection of two putative atypical BTV strains in the Governorate of Gafsa, in the southwest region of Tunisia. Specifically, we recognised the recurrence of an atypical BTV strain (BTV-Y TUN2022) and a novel BTV-W TUN2022.

Prolonged in vitro expansion partially affects phenotypic features and osteogenic potential of ovine amniotic fluid-derived mesenchymal stromal cells.

BACKGROUND AIMS: Ovine amniotic fluid mesenchymal stromal cells (oAFMSCs) are an emerging alternative source of stem cells to develop pre-clinical cell replacement protocols. For tissue engineering purposes, oAFMSCs can be used either immediately after isolation or after in vitro expansion. However, detailed studies are still required to investigate the advantages and drawbacks of their in vitro expansion. METHODS: The phenotype and osteogenic differentiation potential of oAFMSCs were analyzed in relation to in vitro expansion that was carried out for 20 consecutive passages. Expanded oAFMSCs were analyzed for proliferation index, expression profiles of several surface, pluripotency-associated and HLA antigens, global DNA methylation, telomere length and karyotype. The osteogenic differentiation ability of expanded oAFMSCs was assessed by qualitative and quantitative methods. RESULTS: Expanded oAFMSCs reduced their proliferative activity after 10 passages and partially modified the expression of surface antigens and the intracellular distribution of pluripotency-associated markers (NANOG, SOX2 and TERT) after 20 passages. The phenotypic alteration of cultured oAFMSCs was associated with a reduction of in vitro osteogenic plasticity. In detail, after 20 passages of cellular expansion, oAFMSCs lost the ability to increase osteocalcin and decreased collagen type I messenger RNA expression. Also, a lower percentage of cells displayed intracellular calcium release after stimulation with salmon calcitonin. CONCLUSIONS: The results presented here suggest that long-term in vitro expansion may cause significant alterations in phenotypic features and plasticity of oAFMSCs, suggesting a careful re-evaluation of in vitro cultural and temporal conditions before employing expanded oAFMSCs for therapeutic purposes.

One health system supporting surveillance during COVID-19 epidemic in Abruzzo region, southern Italy.

The Istituti Zooprofilattici Sperimentali (IZSs) are public health institutes dealing with the aetiology and pathogenesis of infectious diseases of domestic and wild animals. During Coronavirus Disease 2019 epidemic, the Italian Ministry of Health appointed the IZSs to carry out diagnostic tests for the detection of SARS-CoV-2 in human samples. In particular, the IZS of Abruzzo and Molise (IZS-Teramo) was involved in the diagnosis of SARS-CoV-2 through testing nasopharyngeal swabs by Real Time RT-PCR. Activities and infrastructures were reorganised to the new priorities, in a "One Health" framework, based on interdisciplinary, laboratory promptness, accreditation of the test for the detection of the RNA of SARS-CoV-2 in human samples, and management of confidentiality of sensitive data. The laboratory information system - SILAB - was implemented with a One Health module for managing data of human origin, with tools for the automatic registration of information improving the quality of the data. Moreover, the "National Reference Centre for Whole Genome Sequencing of microbial pathogens - database and bioinformatics analysis" - GENPAT - formally established at the IZS-Teramo, developed bioinformatics workflows and IT dashboard with ad hoc surveillance tools to support the metagenomics-based SARS-CoV-2 surveillance, providing molecular sequencing analysis to quickly intercept the variants circulating in the area. This manuscript describes the One Health system developed by adapting and integrating both SILAB and GENPAT tools for supporting surveillance during COVID-19 epidemic in the Abruzzo region, southern Italy. The developed dashboard permits the health authorities to observe the SARS-CoV-2 spread in the region, and by combining spatio-temporal information with metagenomics provides early evidence for the identification of emerging space-time clusters of variants at the municipality level. The implementation of the One Health module was designed to be easily modelled and adapted for the management of other diseases and future hypothetical events of pandemic nature.

Molecular Characterization of the First African Swine Fever Virus Genotype II Strains Identified from Mainland Italy, 2022.

African swine fever (ASF) is responsible for important socio-economic effects in the global pig industry, especially for countries with large-scale piggery sectors. In January 2022, the African swine fever virus (ASFV) genotype II was identified in a wild boar population in mainland Italy (Piedmont region). This study describes the molecular characterization, by Sanger and next-generation sequencing (NGS), of the first index case 632/AL/2022 and of another isolate (2802/AL/2022) reported in the same month, in close proximity to the first, following multiple ASF outbreaks. Phylogenetic analysis based on the B646L gene and NGS clustered the isolates 632/AL/2022 and 2802/AL/2022 within the wide and most homogeneous p72 genotype II that includes viruses from European and Asian countries. The consensus sequence obtained from the ASFV 2802/AL/2022 isolate was 190,598 nucleotides in length and had a mean GC content of 38.38%. At the whole-genome level, ASF isolate 2802/AL/2022 showed a close genetic correlation with the other representative ASFV genotype II strains isolated between April 2007 and January 2022 from wild and domestic pigs in Eastern/Central European (EU) and Asian countries. CVR subtyping clustered the two Italian ASFV strains within the major CVR variant circulating since the first virus introduction in Georgia in 2007. Intergenic region I73R-I329L subtyping placed the Italian ASFV isolates within the variant identical to the strains frequently identified among wild boars and domestic pigs. Presently, given the high sequence similarity, it is impossible to trace the precise geographic origin of the virus at a country level. Moreover, the full-length sequences available in the NCBI are not completely representative of all affected territories.

Evaluation of next generation sequencing approaches for SARS-CoV-2.

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.

Complete Genome of African Swine Fever Virus Genotype II in Central Italy.

We report here the whole-genome sequence of the African swine fever virus (ASFV) genotype II, strain 20355/RM/2022_Italy, identified in a wild boar in the city of Rome (Lazio region, Italy) in April 2022.

Unveiling the immunomodulatory shift: Epithelial-mesenchymal transition Alters immune mechanisms of amniotic epithelial cells.

Epithelial-mesenchymal transition (EMT) changes cell phenotype by affecting immune properties of amniotic epithelial cells (AECs). The present study shows how the response to lipopolysaccharide of cells collected pre- (eAECs) and post-EMT (mAECs) induces changes in their transcriptomics profile. In fact, eAECs mainly upregulate genes involved in antigen-presenting response, whereas mAECs over-express soluble inflammatory mediator transcripts. Consistently, network analysis identifies CIITA and Nrf2 as main drivers of eAECs and mAECs immune response, respectively. As a consequence, the depletion of CIITA and Nrf2 impairs the ability of eAECs and mAECs to inhibit lymphocyte proliferation or macrophage-dependent IL-6 release, thus confirming their involvement in regulating immune response. Deciphering the mechanisms controlling the immune function of AECs pre- and post-EMT represents a step forward in understanding key physiological events wherein these cells are involved (pregnancy and labor). Moreover, controlling the immunomodulatory properties of eAECs and mAECs may be essential in developing potential strategies for regenerative medicine applications.

Characterization of Epizootic Hemorrhagic Disease Virus Serotype 8 in Naturally Infected Barbary Deer (Cervus elaphus barbarus) and Culicoides (Diptera: Ceratopogonidae) in Tunisia.

Epizootic hemorrhagic disease (EHD) is a Culicoides-borne disease of domestic and wild ruminants caused by EHD virus (EHDV). This virus circulates in multiple serotypes. In late September 2021, a novel strain belonging to EHDV-8 was reported in cattle farms in Central-Western Tunisia, and in the fall of 2022, the same virus was also detected in Italy and Spain. In the present study, we described EHDV-8 occurrence in deer and, a preliminary identification of the potential Culicoides species responsible for virus transmission in selected areas of Tunisia. EHDV-8 was identified in deer carcasses found in 2021 and 2022 in the national reserve of El Feidja, Jendouba, Northwestern Tunisia, and isolated on cell culture. Instead, insect vectors were collected in October 2021 only in the areas surrounding the city of Tozeur (Southern Tunisia) where EHDV-8 cases in cattle were confirmed. Morphological identification showed that 95% of them belonged to the Culicoides kingi and Culicoides oxystoma species and both species tested positive for EHDV-8 RNA. C. imicola was not detected in this collection and EHDV-8 RNA was not evidenced in vector pools collected in 2020, prior to official EHDV-8 emergence. EHDV whole genome sequences were also obtained directly from infected biological samples of deer and positive vectors. EHDV-8 sequences obtained from deer and vectors share a nucleotide identity ranging from 99.42 to 100% and amino acid identity from 99.18 to 100% across all genome segments with the EHDV-8/17 TUN2021 reference sequence.

Spatial and temporal dynamics of West Nile virus between Africa and Europe.

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.

Novel Amplicon-Based Sequencing Approach to West Nile Virus.

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.

Stemness characteristics and osteogenic potential of sheep amniotic epithelial cells.

We set out to characterize stemness properties and osteogenic potential of sheep AEC (amniotic epithelial cells). AEC were isolated from 3-month-old fetuses and expanded in vitro for 12 passages. The morphology, surface markers, stemness markers and osteogenic differentiation were inspected after 1, 6 and 12 passages of expansion, with an average doubling time of 24 h. AEC clearly expressed the stemness markers Oct-3/4 (octamer-binding protein-3/4), Nanog, Sox2 and TERT (telomerase reverse transcriptase) and displayed low levels of global DNA methylation. Culture had moderate effects on cell conditions; some adhesion molecules progressively disappeared from the cell surface, and the expression of Sox2 and TERT was slightly reduced while Nanog increased. No changes occurred in the levels of DNA methylation. Cells organized in 3D spheroids were used for IVD (in vitro differentiation). Within these structures the cells developed a complex intercellular organization that involved extensive intercellular coupling despite continuous cell migration. Marked deposition of calcein in the ECM (extracellular matrix), increased ALP (alkaline phosphatase) activity, expression of bone-related genes (osteocalcin) and the matrix mineralization shown by Alizarin Red staining demonstrate that AEC can undergo rapid and extensive osteogenic differentiation. AEC introduced in experimental bone lesions survived in the site of implantation for 45 days and supported consistent bone neoformation, thus showing promising potential applications in osteogenic regenerative medicine.

Serological, Molecular Prevalence and Genotyping of Coxiella burnetii in Dairy Cattle Herds in Northeastern Algeria.

In Algeria, data on the epidemiology of coxiellosis in cattle are still lacking. In this study, bulk tank milk (BTM) samples from 200 randomly selected dairy cattle herds from Setif province of Algeria were analyzed by an indirect enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Results highlighted that 37% (95% CI: 30.31-43.69%) and 9% (95% CI: 5.03-12.96%) of BTM samples contained Coxiella burnetii antibodies and DNA, respectively. Based on Cohen's kappa coefficient, a very low agreement between the ELISA and PCR results was found (k = 0.0849) (95% CI: 0.00-0.189). For a second experiment, 186 whole blood samples of cows from farms with reproduction disorders were analyzed by molecular tools to detect C. burnetii. This study revealed an overall prevalence of 6.98% (95% CI: 3.32-10.65%). All positive samples determined by conventional PCR were analyzed by real-time quantitative PCR (qPCR). Eleven samples with cycle threshold (Ct) values lower than 35 were selected for genotyping by the multispacer sequence typing (MST) method. The MST12 genotype in BTM samples, the MST32 genotype and a new MST genotype (partial profile) in whole blood samples were identified. Obtained results have allowed us to better understand the epidemiology of bovine coxiellosis in the region of Setif.

West Nile Virus Lineage 2 Overwintering in Italy.

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.

SARS-CoV-2 in Namibian Dogs.

The pandemic of coronavirus disease 19 (COVID-19) has focused the attention of researchers, and especially public opinion, on the role of the human-animal-environment interface in disease emergence. At the beginning of the COVID-19 pandemic, media reports regarding the role of pets in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused significant concern and social anxiety. Although nowadays proven negligible in developed countries, essentially no studies have been performed in low-income African areas where companion animals are often raised differently from high income countries, and the contact patterns occurring in these scenarios could affect the epidemiological scenario. An extensive molecular biology survey was performed from March 2022 to September 2022 on Namibian dogs residing in urban and rural areas, showing a low but not negligible SARS-CoV-2 prevalence (1%; 95CI: 0.33-2.32%) of 5 out of 500. In only one instance (i.e., a 4-year-old female Labrador) was there a clear association that could be established between the infections of the owner and animal. In all other cases, no evidence of human infection could be obtained and no episodes of COVID-19 were reported by the owners. Although no consistent evidence of pet-to-pet transmission was proven in the present study, a cautionary principle suggests intensive and dedicated investigation into companion animal populations, especially when animal contact is frequent and a particularly susceptible population is present.