68 research outputs found
Use of Nanopore Sequencing technology for rapid detection and characterization of pathogens from clinical samples
No full textPrompt and accurate diagnosis is warranted for infectious diseases of domestic animals which may have a significant impact on animal production or clinical practice. In this study, the identification and genetic char- acterization of a bovine enterovirus (BEV) strain isolated from a calf with diarrhea, are described. Two different next generation sequencing platforms were employed. Shotgun metagenomic accomplished by MinION se- quencing (Oxford Nanopore Technologies) allowed the identification of BEV RNA from a cell-culture isolate. BEV was then confirmed by a specific real time RT-PCR assay. To achieve the whole genome of this isolate, sequence reads obtained by MinION were coupled with those originating from NextSeq500 (Illumina). Genomic relatedness and phylogeny with extant BEV strains is also reported. Overall, this manuscript highlights the use of the portable MinION sequence technology as a tool for support diagnostics in veterinary practice
Evolutionary Dinamics of Bluetongue Virus Strains circulating in Italy, 2024-2025
No full textIn 2024, Italy experienced a major resurgence of Bluetongue (BT), a viral disease of ruminants, caused by Bluetongue virus (BTV), an RNA virus circulating in multiple serotypes transmitted by Culicoides midges. By April 2025, over 7,000 confirmed cases were reported, mainly involving BTV-3, BTV-4, and BTV-8. We analyzed the full genomes of BTV strains collected from different Italian regions in 2024 through WGS and phylogenetic analysis. At least three distinct genomic constellations of BTV-3 were identified, two resulting from multiple reassortments involving European strains, and one closely related to the Northern European BTV-3. BTV-4 strains also displayed multiple genomic profiles, with gene segments from different origins. BTV-8 showed high genetic identity (up to 100% in Seg-2) with strains from France, suggesting viral spread through the trade of viremic animals. Notably, BTV-8 genome segments were found within BTV-3 and BTV-4 strains, highlighting the ongoing role of reassortment in shaping BTV evolution. These findings reveal the complex genomic landscape of BTV in Italy and emphasize the importance of WGS in tracking viral dynamics
Outbreak of porcine epidemic diarrhoea virus (PEDV) in Abruzzi region, central-Italy
No full textHere we report and characterize a porcine epidemic diarrhea (PED) outbreak which occurred in a swine fattening farm in the province of Teramo, Abruzzi region (central Italy), in January 2016. PED virus (PEDV) identification was determined by real-time RT-PCR performed on RNAs purified from fecal samples collected from two symptomatic pigs. Whole genome sequence (PEDV 1842/2016) was also obtained by next generation sequencing straight from RNA purified from one fecal sample. Genome comparison with extant global PEDV strains revealed a high nucleotide identity with recently reported European and American S-INDEL PEDVs. Efficient sequencing, share of genomic data combined with the implementation of epidemiological tools would be the ideal approach for study and analysis of transboundary infectious diseases as PED
Detection of vesivirus in minks (Neovison vison), Italy 2021
No full textVesiviruses 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
Genome characterization of feline morbillivirus from Italy
No full textFeline morbillivirus (FeMV) has been recently identified by RT-PCR in the urine sample of a nephropathic cat in Italy. In this report, we describe the whole genome sequence of strain Piuma/2015 obtained by combination of sequence independent single primer amplification method (SISPA) and next generation sequencing (NGS) starting from RNA purified from the infected urine sample. The existence in Germany and Turkey of FeMVs from cats divergent from Piuma/2015, suggests the presence of FeMV heterogeneity in Europe as it has been described previously in Japan and China
First report of feline morbillivirus in Europe
No full textFeline morbillivirus was detected in urine samples of a 15 year old cat suffering from severe
nephropathy. Viral RNA was not detected in blood and faecal samples and also the most
common pathogens associated to cat kidney failure were not found. This report describes
the first evidence of feline morbillivirus in Europe
First Detection of West Nile Virus by Nasopharyngeal Swab, Followed by Phylogenetic Analysis
No full textWest Nile Virus, an arthropod-borne RNA virus, may result in severe neurological disease. West Nile neuroinvasive disease is characterized by meningitis, encephalitis, and possible acute flaccid paralysis. Here, we report a case of neuroinvasive WNV in a 65-year-old woman hospitalized for hyperpyrexia, chills, intense asthenia, and continuous vomiting. Within days, her clinical condition worsened with the onset of severe neurological symptoms, leading to her death within 10 days despite supportive therapies being administered. The diagnosis of West Nile disease was made through nucleic acid amplification testing (NAAT) on blood and cerebrospinal fluid. However, in the final stages of the illness, cerebrospinal fluid collection was not possible due to the patient’s critical condition, and a nasopharyngeal swab was used instead. The nasopharyngeal swab facilitated the collection of a sample, which was subsequently analyzed for the presence of the virus and allowed for sequencing, showing that it was a strain that had been circulating in Sardinia for some time and had demonstrated its pathogenicity by causing the death of a hawk in 2021. This case report highlights the rapid progression and severity of WNV infection, particularly in vulnerable individuals, and suggests the potential utility of nasopharyngeal swabs as a less invasive option for sample collection. It also underscores the potential for the zoonotic transmission of the virus from birds to humans through vectors, emphasizing the importance of monitoring and controlling WNV outbreaks, especially in regions where such circulation is observed
Complete sequence of human mitochondrial DNA obtained by combining multiple displacement amplification and next-generation sequencing on a single oocyte
No full textMitochondrial DNA (mtDNA) plays a key role in the development of a competent oocyte. In this study, the complete mtDNA sequence obtained for the first time by multiple displacement amplification approach in combination with next-generation sequencing from a single human oocyte is reported (GenBank accession no. KT364276). The analysis of oocyte mitochondria' mutations could provide a better understanding of the genetic variants correlated with the oocyte quality
Mitochondrial heteroplasmy profiling in single human oocytes by next-generation sequencing
No full textMitochondrial DNA (mtDNA) plays a key role in the development of a competent oocyte. Mutations of
the mitochondrial genome lead to an altered energetic metabolism with negative effects on oocyte
developmental competence. In this study, mtDNA heteroplasmy at an intra-oocyte level and between
the different analyzed human oocytes (n1⁄412) was identified by a next-generation sequencing (NGS)
protocol previously developed by this research group and submitted to GenBank. This method highlighted,
in particular, variants in the genes involved in the respiratory chain providing a direct indication
of the cell-specific damage within the mitochondrial genome as predictor of the oocyte quality
Profiling of mitochondrial heteroplasmy in single human oocytes by next-generation sequencing
No full textMitochondrial DNA (mtDNA) plays a crucial role in the development of a competent oocyte. Indeed, mtDNA alterations may predispose to chromosome nondisjunction, resulting in infertility due to a reduced vitality and quality of oocytes and embryos. In this methods paper, the multiple displacement amplification approach was applied in combination with next-generation sequencing (NGS) to amplify and sequence, in single-end, the entire mtDNA of single human oocytes to directly construct genomic NGS libraries, and subsequently, to highlight and quantify the mutations they presented. The bioinformatic workflow was carried out with a specific ad hoc developed in-house software. This approach proved to be sensitive and specific, also highlighting the mutations present in heteroplasmy, showing deletion, insertion or substitution mutations in the genes involved in the respiratory chain, even if the found variants were benign or of uncertain meaning. The analysis of mtDNA mutations in the oocyte could provide a better understanding of specific genetic abnormalities and of their possible effect on oocyte developmental competence. This study shows how this approach, based on a massive parallel sequencing of clonally amplified DNA molecules, allows to sequence the entire mitochondrial genome of single oocytes in a short time and with a single analytical run and to verify mtDNA mutations
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