African swine fever (ASF) poses a severe threat to the global pig industry for which currently there is no available vaccine. The aetiological ASF virus (ASFV) has a predilection for cells of the myeloid lineage, however little is known about its interaction with polarised macrophages. This study focused on the in vitro interactions of porcine monocyte-derived un-activated (moMΦ), classically (moM1), alternatively (moM2), and IFN-α-activated macrophages with two genotype I ASFV strains: virulent 22653/14 and attenuated NH/P68. At a high multiplicity of infection, NH/P68, but not 22653/14, presented a reduced ability to infect moM1 and IFN−α-activated moMΦ compared to moMΦ. IFN-α activation resulted in a dose-dependent reduction in the proportion of ASFV-infected cells. Both strains replicated efficiently in all the subsets. While higher levels of IL-1α, IL-1β, and IL-18 were secreted by NH/P68-infected moM1 compared to 22653/14, both strains negatively affected moMΦ ability to release IL-6, IL-12, TNF-α in response to classical activation or stimulation with a TLR2 agonist. Our results suggest that ASFV 22653/14 covertly replicates in macrophages, compromising the development of effective immune responses. Attenuated NH/P68 has partially lost these mechanisms, which may enhance immune surveillance. A better understating of these mechanisms should aid the rational design of live attenuated ASFV vaccines.

Macrophages are phagocytic cells involved in maintaining tissue homeostasis and defense against pathogens. Macrophages may be polarized into different functionally specialized subsets. M2c macrophages arise following stimulation with IL-10 or TGF-β and mediate anti-inflammatory and tissue repair functions. M2c macrophages remain poorly characterized in the pig, thus we investigated the impact of these regulatory cytokines on porcine monocyte-derived macrophages (moMΦ). The phenotype and functionality of these cells was characterized though confocal microscopy, flow cytometry, ELISA, and RT-qPCR. Both cytokines induced CD14 and MHC II DR down-regulation and reduced IL-6, TNF-α, and CD14 expression, suggestive of an anti-inflammatory phenotype. Interestingly, neither IL-10 or TGF-β were able to trigger IL-10 induction or release by moMΦ. Differences between these cytokines were observed: stimulation with IL-10, but not TGF-β, induced up-regulation of both CD16 and CD163 on moMΦ. In addition, IL-10 down-regulated expression of IL-1β and IL-12p40 4h post-stimulation and induced a stronger impairment of moMΦ ability to respond to either TLR2 or TLR4 agonists. Overall, our results provide an overview of porcine macrophage polarization by two immunosuppressive cytokines, revealing differences between IL-10 and TGF-β, and reporting some peculiarity of swine, which should be considered in translational studies.

African Swine Fever Virus (ASFV) has tropism for macrophages, which seems to play a crucial role in disease pathogenesis and viral dissemination. Previous studies showed that ASFV developed mechanisms to evade type I interferon (IFN) responses. Hence, we analyzed the ability of ASFV strains of diverse virulence to modulate IFN-β and IFN-α responses. Porcine monocyte-derived macrophages un-activated (moMΦ) or activated with IFN-α (moMΦ + FN-α) were infected with virulent (22653/14) or attenuated (NH/P68) ASFV strains, and expressions of IFN-β and of 17 IFN-α subtypes genes were monitored over time. ASFV strains of diverse virulence induced different panels of IFN genes: infection of moMΦ with either strains caused statistically significant up-regulation of IFN-α3, -α7/11, whereas only attenuated NH/P68 determined statistically significant up-regulation of IFN-α10, -α12, -α13, -α15, -α17, and IFN-β. Infection of activated moMΦ with either strains resulted in up-regulation of IFN-β and many IFN-α subtypes, but statistical significance was found only for IFN-α1, -α10, -α15, -α16, -α17 in response to NH/P68-infection only. These data revealed differences in type I IFNs expression patterns, with differences between strains of diverse virulence. In addition, virulent 22653/14 ASFV seems to have developed mechanisms to suppress the induction of several type I IFN genes.

Porcine circovirus 3 (PCV3) is a recently discovered member of the Circoviridae family. So far, its presence has been reported in North America, Asia, South America, and Europe. In this study, blood and tissue samples from 189 Sardinian suids (34 domestic pigs, 115 feral free ranging pigs, and 39 wild boars) were used to genetically characterize the PCV3 strains from Sardinia. PCV3 infection in the animals was confirmed by real time PCR. The detection rate in the three groups analyzed was l7.64% in domestic pigs, 77.39% in free ranging pigs, and 61.54% in wild boars. Moreover, our results showed that co-infection of PCV3 with other viruses is quite a common occurrence. Molecular characterization of Sardinian PCV3 strains was performed by sequencing 6 complete genomes and 12 complete cap genes. Our results revealed that there is a high similarity between our strains and those identified in different countries, confirming the genetic stability of PCV3 regardless of geographical origin. Haplotype network analysis revealed the presence of 6 whole genomes or 12 unique ORF2 haplotypes and a nonsynonymous mutation in ORF2 that leads to an R14K amino acid substitution. Phylogenetic analysis of whole genome and ORF2 was also conducted. The Sardinian strains were allocated in three different clusters of phylogenetic trees of both complete genome and ORF2. With this study, we have provided a snapshot of PCV3 circulation in Sardinia. Our findings might help to achieve a deeper understanding of this emerging porcine virus.

African swine fever virus (ASFV), the cause of a devastating disease affecting domestic and wild pigs, has been present in Sardinia since 1978. In the framework of the regional ASF eradication plan, 4484 illegal pigs were culled between December 2017 and February 2020. The highest disease prevalence was observed in the municipality with the highest free-ranging pig density, and culling actions drastically reduced ASFV circulation among these animals. ASFV-antibody were detected in 36.7% of tested animals, which were apparently healthy, thus, the circulation of low-virulence ASFV isolates was hypothesized. ASFV genome was detected in 53 out of 2726 tested animals, and virus isolation was achieved in two distinct culling actions. Two ASFV haemadsorbing strains were isolated from antibody-positive apparently healthy pigs: 55234/18 and 103917/18. Typing analysis revealed that both isolates belong to p72 genotype I, B602L subgroup X; phylogenetic analysis based on whole genome sequencing data showed that they were closely related to Sardinian ASFV strains collected since 2010, especially 22653/Ca/2014. Our data suggested the absence of immune-escaped ASFV variants circulating among free-ranging pigs, indicating that other elements contributed to virus circulation among these animals. Understanding factors behind disease persistence in endemic settings might contribute to developing effective countermeasures against this disease.

African swine fever virus (ASFV) is the etiological agent of the devastating disease African swine fever (ASF), for which there is currently no licensed vaccine or treatment available. ASF is defined as one of the most serious animal diseases identified to date, due to its global spread in regions of Africa, Europe and Asia, causing massive economic losses. On the Italian island of Sardinia, the disease has been endemic since 1978, although the last control measures put in place achieved a significant reduction in ASF, and the virus has been absent from circulation since April 2019. Like many large DNA viruses, ASFV mutates at a relatively slow rate. However, the limited availability of whole-genome sequences from spatial-localized outbreaks makes it difficult to explore the small-scale genetic structure of these ASFV outbreaks. It is also unclear if the genetic variability within outbreaks can be captured in a handful of sequences, or if larger sequencing efforts can improve phylogenetic reconstruction and evolutionary or epidemiological inference. The aim of this study was to investigate the phylogenetic patterns of ASFV outbreaks between 1978 and 2018 in Sardinia, in order to characterize the epidemiological dynamics of the viral strains circulating in this Mediterranean island. To reach this goal, 58 new whole genomes of ASFV isolates were obtained, which represents the largest ASFV whole-genome sequencing effort to date. We provided a complete description of the genomic diversity of ASFV in terms of nucleotide mutations and small and large indels among the isolates collected during the outbreaks. The new sequences capture more than twice the genomic and phylogenetic diversity of all the previously published Sardinian sequences. The extra genomic diversity increases the resolution of the phylogenetic reconstruction, enabling us to dissect, for the first time, the genetic substructure of the outbreak. We found multiple ASFV subclusters within the phylogeny of the Sardinian epidemic, some of which coexisted in space and time.