Small Ruminant Lentiviruses (SRLV) include at least 4 viral highly divergent genotypes. Genotypes A and B are widely distributed and genotypes C and E have been recognized in restricted geographic areas. New phylogroups have been identified targeting conserved regions. However, this approach suffers from the potential risk to misamplify highly divergent strains. Pathogenic strains are easily adapted to fibroblastic cells, but non-pathogenic strains isolation may require a different approach. We developed a fast and effective method for SRLV full genome characterization after cell culture isolation. Spleen samples were collected during regular slaughter from sheep and goats in northwestern Italy. Spleen-derived macrophage cultures were monitored for reverse transcriptase activity and RNA was extracted from the supernatant of positive cultures. Using Illumina MiSeq platform 22 new full genome sequences were obtained. The success of this approach is based on the following features: spleen is one of the main target for SRLV persistence; red pulp is a reserve of resident macrophages, the main target for SRLV replication in vivo; RTA is a sensitive assay for any replicating retrovirus; de novo sequencing do not require genetic knowledge in advance.

Background: Mycoplasmas are the simplest bacteria capable of autonomous replication. Their evolution proceeded from gram-positive bacteria, with the loss of many biosynthetic pathways and of the cell wall. In this work, the liposoluble protein complement of Mycoplasma agalactiae, a minimal bacterial pathogen causing mastitis, polyarthritis, keratoconjunctivitis, and abortion in small ruminants, was subjected to systematic characterization in order to gain insights into its membrane proteome composition. Results: The selective enrichment for M. agalactiae PG2^T liposoluble proteins was accomplished by means of Triton X-114 fractionation. Liposoluble proteins were subjected to 2-D PAGE-MS, leading to the identification of 40 unique proteins and to the generation of a reference 2D map of the M. agalactiae liposoluble proteome. Liposoluble proteins from the type strain PG2 and two field isolates were then compared by means of 2D DIGE, revealing reproducible differences in protein expression among isolates. An in-depth analysis was then performed by GeLC-MS/MS in order to achieve a higher coverage of the liposoluble proteome. Using this approach, a total of 194 unique proteins were identified, corresponding to 26% of all M. agalactiae PG2^T genes. A gene ontology analysis and classification for localization and function was also carried out on all protein identifications. Interestingly, the 11.5% of expressed membrane proteins derived from putative horizontal gene transfer events. Conclusions: This study led to the in-depth systematic characterization of the M. agalactiae liposoluble protein component, providing useful insights into its membrane organization.

In this study the enzymatic activity of Mycoplasma agalactiae MAG_5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG_5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG_5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl₂ at temperatures ranging from 37 to 45°C. According to in silico analyses, the position of the gene encoding MAG_5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG_5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants.

Coronaviruses are known to be harmful and heterogeneous viruses, able to infect a large number of hosts. Among them, SADS-CoV (Swine Acute Diarrhea Syndrome Coronavirus), also known as PEAV (Porcine Enteric Alphacoronavirus), or SeA-CoV (Swine Enteric Alphacoronavirus), is the most recent Alphacoronavirus discovered, and caused several outbreaks reported in Chinese swine herds between late 2016 and 2019. We performed an upgraded phylodinamic reconstruction of SADS-CoV based on all whole genomes available on 21 June 2021. Results showed a very close relationship between SADS-CoV and HKU2-like CoV, which may represent the evolutionary intermediate step towards the present SADS-CoV. The direct progenitor of SADS-CoV is so far unknown and, although it is well known that horseshoe bats are reservoirs for Rhinolophus bat coronavirus HKU2-like (HKU2-like CoVs), the transmission path from bats to pigs is still unclear. The discrepancies in the phylogenetic position of rodent CoV, when different molecular markers were considered, corroborate the recombination hypothesis, suggesting that wild rats, which are frequent in farms, may have played a key role. The failure of the attempt at molecular dating, due to the lack of a clock signal, also corroborates the occurrence of a recombination event hypothesis. Zoonotic infections originating in wildlife can easily become a significant threat for human health. In such a context, due to the high recombination and cross-species capabilities of Coronavirus, SADS-CoV represents a possible high-risk pathogen for humans which needs a constant molecular monitoring.