In the last decade, an upsurge of human leishmaniasis has been reported in the Emilia-Romagna region, Northeast Italy. Epidemiologic data have raised doubts about the role of dogs as the main reservoirs for Leishmania infantum. In the present study, a total of 1,077 wild animals were screened for L. infantum DNA in earlobe and spleen samples from 2019 to 2022. The lymph nodes were tested only in 23 animals already positive in the earlobe and/or spleen. A total of 71 (6.6%) animals resulted positive in at least one of the sampled tissues, including 3/18 (16.7%) wolves, 6/39 (15.4%) European hares, 38/309 (12.3%) roe deer, 1/11 (9.1%) red deer, 8/146 (4.9%) wild boars, 13/319 (4.1%) red foxes, 1/54 (1.9%) porcupine, and 1/59 (1.7%) European badger. Most of the infected animals (62/71) tested positive only in the earlobe tissue, only four animals (two roe deer and two wild boars) tested positive only in the spleen, and five animals (three roe deer and two red foxes) resulted positive for both tissues. L. infantum DNA was detected in the lymph nodes of 6/23 animals. L. infantum detection occurred in all seasons associated with low real-time PCR Ct values. Further research is needed in order to clarify the role of wildlife in the re-emerging focus of leishmaniasis in Northeast Italy.

Parvovirus B19 (B19V), an ssDNA virus in the family Parvoviridae, is a human pathogenic virus, responsible for a wide range of clinical manifestations, still in need of effective and specific antivirals. DNA structures, including G-quadruplex (G4), have been recognised as relevant functional features in viral genomes, and small-molecule ligands binding to these structures are promising antiviral compounds. Bioinformatic tools predict the presence of potential G4 forming sequences (PQSs) in the genome of B19V, raising interest as targets for antiviral strategies. Predictions locate PQSs in the genomic terminal regions, in proximity to replicative origins. The actual propensity of these PQSs to form G4 structures was investigated by circular dichroism spectroscopic analysis on synthetic oligonucleotides of corresponding sequences. No signature of G4 structures was detected, and the interaction with the G4 ligand BRACO-19 (N,N′-(9-{[4-(dimethylamino)phenyl]amino}acridine-3,6-diyl)bis(3-pyrrolidin-1-ylpropanamide) did not appear consistent with the stabilisation of G4 structures. Any potential role of PQSs in the viral lifecycle was then assessed in an in vitro infection model system, by evaluating any variation in replication or expression of B19V in the presence of the G4 ligands BRACO-19 and pyridostatin. Neither showed a significant inhibitory activity on B19V replication or expression. Experimental challenge did not support bioinformatic predictions. The terminal regions of B19V are characterised by relevant sequence and symmetry constraints, which are functional to viral replication. Our experiments suggest that these impose a stringent requirement prevailing over the propensity of forming actual G4 structures.

Parvovirus B19 (B19V) is a human pathogenic virus of clinical relevance, characterized by a selective tropism for erythroid progenitor cells in bone marrow. Relevant information on viral characteristics and lifecycle can be obtained from experiments involving engineered genetic systems in appropriate in vitro cellular models. Previously, a B19V genome of defined consensus sequence was designed, synthesized and cloned in a complete and functional form, able to replicate and produce infectious viral particles in a producer/amplifier cell system. Based on such a system, we have now designed and produced a derived B19V minigenome, reduced to a replicon unit. The genome terminal regions were maintained in a form able to sustain viral replication, while the internal region was clipped to include only the left-side genetic set, containing the coding sequence for the functional NS1 protein. Following transfection in UT7/EpoS1 cells, this minigenome still proved competent for replication, transcription and production of NS1 protein. Further, the B19V minigenome was able to complement B19-derived, NS1-defective genomes, restoring their ability to express viral capsid proteins. The B19V genome was thus engineered to yield a two-component system, with complementing functions, providing a valuable tool for studying viral expression and genetics, suitable to further engineering for purposes of translational research.

In the last decade, an upsurge of human leishmaniasis has been reported in the Emilia-Romagna region, Northeast Italy. Epidemiologic data raised doubts about the role of dogs as the main reservoirs for Leishmania infantum. In the present study, a total of 1,077 wild animals were screened for L. infantum DNA in earlobe and spleen samples from 2019 to 2022. The lymph nodes were tested only in the 23 animals already positive in the earlobe and/or spleen. A total of 71 (6.6%) animals resulted positive in at least one of the sampled tissues, including 3/18 (16.7%) wolves, 6/39 (15.4%) European hares, 38/309 (12.3%) roe deer, 1/11 (9.1%) red deer, 8 (4.9%) wild boars, 13/319 (4.1%) red foxes, 1/54 (1.9%) porcupine, and 1/59 (1.7%) European badger. Most of the infected animals (62/71) tested positive only in the earlobe tissue, just 4 animals (2 roe deer and 2 wild boars) tested positive only in the spleen, and 5 animals (3 roe deer and 2 red foxes) resulted positive for both tissues. L. infantum DNA was detected in the lymph nodes of 6/23 ani-mals. L. infantum detection occurred in all seasons associated with low real-time PCR Ct values. Further research is needed in order to clarify the role of wildlife in the re-emerging focus of leishmaniasis in Northeast Italy.