Analysis of Genomic Characteristics of SARS-CoV-2 in Italy, 29 January to 27 March 2020.

We performed next-generation sequencing (NGS), phylogenetic analysis, gene flows, and N- and O-glycosylation prediction on SARS-CoV-2 genomes collected from lab-confirmed cases from different Italian regions. To this end, a total of 111 SARS-CoV-2 genomes collected in Italy between 29 January and 27 March 2020 were investigated. The majority of the genomes belonged to lineage B.1, with some descendant lineages. The gene flow analysis showed that the spread occurred mainly from the north to the center and to the south of Italy, as confirmed by epidemiological data. The mean evolutionary rate estimated here was 8.731 × 10-4 (95% highest posterior density, HPD intervals 5.809 × 10-4 to 1.19 × 10-3), in line with values reported by other authors. The dated phylogeny suggested that SARS-CoV-2 lineage B.1 probably entered Italy between the end of January and early February 2020. Continuous molecular surveillance is needed to trace virus circulation and evolution.

Clostridium botulinum group I strain genotyping by 15-locus multilocus variable-number tandem-repeat analysis.

Clostridium botulinum is a taxonomic designation that encompasses a broad variety of spore-forming, Gram-positive bacteria producing the botulinum neurotoxin (BoNT). C. botulinum is the etiologic agent of botulism, a rare but severe neuroparalytic disease. Fine-resolution genetic characterization of C. botulinum isolates of any BoNT type is relevant for both epidemiological studies and forensic microbiology. A 10-locus multiple-locus variable-number tandem-repeat analysis (MLVA) was previously applied to isolates of C. botulinum type A. The present study includes five additional loci designed to better address proteolytic B and F serotypes. We investigated 79 C. botulinum group I strains isolated from human and food samples in several European countries, including types A (28), B (36), AB (4), and F (11) strains, and 5 nontoxic Clostridium sporogenes. Additional data were deduced from in silico analysis of 10 available fully sequenced genomes. This 15-locus MLVA (MLVA-15) scheme identified 86 distinct genotypes that clustered consistently with the results of amplified fragment length polymorphism (AFLP) and MLVA genotyping in previous reports. An MLVA-7 scheme, a subset of the MLVA-15, performed on a lab-on-a-chip device using a nonfluorescent subset of primers, is also proposed as a first-line assay. The phylogenetic grouping obtained with the MLVA-7 does not differ significantly from that generated by the MLVA-15. To our knowledge, this report is the first to analyze genetic variability among all of the C. botulinum group I serotypes by MLVA. Our data provide new insights into the genetic variability of group I C. botulinum isolates worldwide and demonstrate that this group is genetically highly diverse.

Extensive Genome Exploration of Clostridium botulinum Group III Field Strains.

In animals, botulism is commonly sustained by botulinum neurotoxin C, D or their mosaic variants, which are produced by anaerobic bacteria included in Clostridium botulinum group III. In this study, a WGS has been applied to a large collection of C. botulinum group III field strains in order to expand the knowledge on these BoNT-producing Clostridia and to evaluate the potentiality of this method for epidemiological investigations. Sixty field strains were submitted to WGS, and the results were analyzed with respect to epidemiological information and compared to published sequences. The strains were isolated from biological or environmental samples collected in animal botulism outbreaks which occurred in Italy from 2007 to 2016. The new sequenced strains belonged to subspecific groups, some of which were already defined, while others were newly characterized, peculiar to Italian strains and contained genomic features not yet observed. This included, in particular, two new flicC types (VI and VII) and new plasmids which widen the known plasmidome of the species. The extensive genome exploration shown in this study improves the C. botulinum and related species classification scheme, enriching it with new strains of rare genotypes and permitting the highest grade of discrimination among strains for forensic and epidemiological applications.

Identification and characterization of SARS-CoV-2 clusters in the EU/EEA in the first pandemic wave: additional elements to trace the route of the virus.

A high-quality dataset of 3289 complete SARS-CoV-2 genomes collected in Europe and European Economic Area (EAA) in the early phase of the first wave of the pandemic was analyzed. Among all single nucleotide mutations, 41 had a frequency ≥ 1%, and the phylogenetic analysis showed at least 6 clusters with a specific mutational profile. These clusters were differentially distributed in the EU/EEA, showing a statistically significant association with the geographic origin. The analysis highlighted that the mutations C14408T and C14805T played an important role in clusters selection and further virus spread. Moreover, the molecular analysis suggests that the SARS-CoV-2 strain responsible for the first Italian confirmed COVID-19 case was already circulating outside the country.

Multiple-locus variable number of tandem repeat analysis as a tool for molecular epidemiology of botulism: The Italian experience.

Clostridium botulinum is the bacterial agent of botulism, a rare but severe neuro-paralytic disease. Because of its high impact, in Italy botulism is monitored by an ad hoc surveillance system. The National Reference Centre for Botulism, as part of this system, collects and analyzes all demographic, epidemiologic, microbiological, and molecular data recovered during cases and/or outbreaks occurred in Italy. A panel of 312 C. botulinum strains belonging to group I were submitted to MLVA sub-typing. Strains, isolated from clinical specimens, food and environmental samples collected during the surveillance activities, were representative of all forms of botulism from all Italian regions. Through clustering analysis isolates were grouped into 12 main clusters. No regional or temporal clustering was detected, demonstrating the high heterogeneity of strains circulating in Italy. This study confirmed that MLVA is capable of sub-typing C. botulinum strains. Moreover, MLVA is effective at tracing and tracking the source of contamination and is helpful for the surveillance system in terms of planning and upgrading of procedures, activities and data collection forms.

Genomic characterization of Italian Clostridium botulinum group I strains.

Clostridium botulinum is a gram-positive bacterium capable of producing the botulinum neurotoxin, a powerful poison that causes botulism, a severe neuroparalytic disease. Its genome has been sequenced entirely and its gene content has been analyzed. To date, 19 full genomes and 64 draft genomes are available. The geographical origin of these genomes is predominantly from the US. In the present study, 10 Italian genomes of C. botulinum group I were analyzed and compared with previously sequenced group I genomes, in order to genetically characterize the Italian population of C. botulinum group I and to investigate the phylogenetic relationships among different lineages. Using the suites of software ClonalFrame and ClonalOrigin to perform genomic analysis, we demonstrated that Italian C. botulinum group I population is phylogenetically heterogeneous encompassing different and distant lineages including overseas strains, too. Moreover, a high recombination rate was demonstrated in the evolution of C. botulinum group I species. Finally, genome sequencing of the strain 357 led us to identify a novel botulinum neurotoxin subtype, F8.

Draft Genome Sequence of Clostridium botulinum B2 450 Strain from Wound Botulism in a Drug User in Italy.

Here, we report the draft genome sequence of Clostridium botulinum B2 450, responsible for the first reported case of wound botulism in a drug user in Italy.

Whole-Genome Sequence of Clostridium botulinum A2B3 87, a Highly Virulent Strain Involved in a Fatal Case of Foodborne Botulism in Italy.

Here, we report the genome sequence of a rare bivalent strain of Clostridium botulinum, A2B3 87. The strain was isolated from a foodborne botulism case that occurred in Italy in 1995. The case was characterized by rapid evolution of the illness and failure of conventional treatments.

Metabotropic glutamate receptors 5 blockade reverses spatial memory deficits in a mouse model of Parkinson's disease.

Visuo-spatial deficits are the most consistently reported cognitive abnormalities in Parkinson's disease (PD), and they are frequently associated to motor symptoms in the early stages of the disease when dopamine loss is moderate and still restricted to the caudate-putamen. The metabotropic glutamate receptor 5 (mGluR5) antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), has beneficial effects on motor symptoms in animal models of PD. However, the effects of MPEP on the cognitive deficits of the disease have never been investigated. Thus, the purpose of this study was to explore its therapeutic potentials by investigating its effects on the visuo-spatial deficits induced by 6-hydroxydopamine (6-OHDA) lesions of dorsal striatum in CD1 mice. The results demonstrated that systemic injections of MPEP (6, 12, and 24 mg/kg, i.p.) impair visuo-spatial discrimination in intact mice at high concentrations, whereas lower doses (1.5 and 3 mg/kg, i.p.) were void of effects. Nevertheless, when an ineffective dose (MPEP 3 mg/kg) was injected, either acutely or subchronically (8 days), it antagonized the visuo-spatial discrimination deficit induced by bilateral dopamine lesion of the striatum. Furthermore, the same treatment increased contralateral turning induced by L-DOPA in mice bearing unilateral 6-OHDA lesion. These results confirm the therapeutic potential of mGluR5 blockade on motor symptoms induced by reduced striatal dopamine function. Further, they demonstrate that mGluR5 blockade may also have beneficial effects on cognitive deficits induced by dopamine depletion.

PrPSc in salivary glands of scrapie-affected sheep.

The salivary glands of scrapie-affected sheep and healthy controls were investigated for the presence of the pathological prion protein (PrP(Sc)). PrP(Sc) was detected in major (parotid and mandibular) and minor (buccal, labial, and palatine) salivary glands of naturally and experimentally infected sheep. Using Western blotting, the PrP(Sc) concentration in glands was estimated to be 0.02 to 0.005% of that in brain. Immunohistochemistry revealed intracellular depositions of PrP(Sc) in ductal and acinar epithelia and occasional labeling in the lumina of salivary ducts. The presence of PrP(Sc) in salivary glands highlights the possible role of saliva in the horizontal transmission of scrapie.

Prion protein alleles showing a protective effect on the susceptibility of sheep to scrapie and bovine spongiform encephalopathy.

The susceptibility of sheep to classical scrapie and bovine spongiform encephalopathy (BSE) is mainly influenced by prion protein (PrP) polymorphisms A136V, R154H, and Q171R, with the ARR allele associated with significantly decreased susceptibility. Here we report the protective effect of the amino acid substitution M137T, I142K, or N176K on the ARQ allele in sheep experimentally challenged with either scrapie or BSE. Such observations suggest the existence of additional PrP alleles that significantly decrease the susceptibility of sheep to transmissible spongiform encephalopathies, which may have important implications for disease eradication strategies.