MicroRNAs (miRNAs) represent a family of small non-coding ribonucleic acids that post-transcriptionally inhibits the expression of their target messenger RNAs (mRNAs), thereby acting as general gene repressors. In this study we examined the relative quantity and stability of miRNA subjected to a long period of freezing; we compared the stability of eight miRNAs in the plasma of five human healthy controls before freezing and after six and 12 months of storage at −80 °C. In addition, we examined the plasma frozen for 14 years and the amount of miRNA still available. Using a Life Technologies protocol to amplify and quantify plasma miRNAs from EDTA (Ethylene Diamine Tetraacetic Acid)-treated blood, we analyzed the stability of eight miRNAs, (miR-125b-5p, miR-425-5p, miR-200b-5p, miR-200c-3p, miR-579-3p, miR-212-3p, miR-126-3p, and miR-21-5p). The miRNAs analyzed showed a high stability and long frozen half-life.

African swine fever virus (ASFV) is the aetiological agent of a highly lethal haemorrhagic disease affecting pigs that inflicts significant economic damage on the swine industry. ASF is present in many African countries, in several eastern and central European countries and in Sardinia (Italy). Sequence analyses of variable genomic regions have been extensively used for molecular epidemiological studies of ASFV isolates. Previous sequencing data of genes that codify for viral protein p54, p72 and the central variable region (CVR) within the B602L gene revealed that Sardinian isolates show a very low level of variability. To achieve a finer level of discrimination among such closely related viruses, in this study, we have chosen three different genome regions to investigate the within-genotype relationships and to provide a more accurate assessment of the origin of outbreaks. The analysis of p30 and I73R/I329L sequences obtained from ASFV collected in Sardinia over a 13-years period confirms a remarkable genetic stability in these regions. The sequence comparison of the protein encoded by the EP402R gene (CD2v), carried out on various strains from 1978 to 2014, revealed a temporal subdivision of Sardinian viruses into two subgroups: one group includes the historical isolates from 1978 to 1990, and the second one is comprised of the viruses collected from 1990 until 2014. These data, together with those obtained from CVR within the B602L gene analysis, demonstrated that the viruses circulating in Sardinia belong to p72 genotype I, but have undergone genetic variations in two different regions of the genome since 1990. We proposed the cytoplasmic region of CD2v protein as a new genetic marker that could be use to analyse ASFVs from different locations to track virus spread. Our study reaffirms the need to analyse other genome regions in order to improve the molecular characterization of ASFV.

African swine fever (ASF) is a devastating disease for which there is no vaccine available. The ASF virus (ASFV) primarily infects cells of the myeloid lineage and this tropism is thought to be crucial for disease pathogenesis. A detailed in vitro characterization of the interactions of a virulent Sardinian isolate (22653/14) and a tissue culture adapted avirulent strain (BA71V) of ASFV with porcine monocytes, un-activated (moMΦ), classically (moM1) and alternatively (moM2) activated monocyte-derived macrophages was conducted in an attempt to better understand this relationship. Using a multiplicity-of-infection (MOI) of 1, both viruses were able to infect monocytes and macrophage subsets, but BA71V presented a reduced ability to infect moM1 compared to 22653/14, with higher expression of early compared to late proteins. Using an MOI of 0.01, only 22653/14 was able to replicate in all the macrophage subsets, with initially lowest in moM1 and moM2. No differences were observed in the expression of CD163 between ASFV infected and uninfected bystander cells. ASFV down-regulated CD16 expression but did not modulate MHC class II levels in monocytes and macrophage subsets. BA71V-infected but not 22653/14-infected moMΦ and moM2 presented with a reduced expression of MHC class I compared to the mock-infected controls. Higher levels of IL-18, IL1-β and IL-1α were released from moM1 after infection with BA71V compared to 22653/14 or mock-infected control. These results revealed differences between these ASFV strains, suggesting that virulent isolates have evolved mechanisms to counteract activated macrophages responses, promoting their survival, dissemination in the host and so ASF pathogenesis.

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.

African swine fever (ASF) is a devastating disease for which there is no vaccine. The ASF virus (ASFV) can infect dendritic cell (DC), but despite the critical role these cells play in induction of adaptive immunity, few studies investigated their response to ASFV infection. We characterized the in vitro interactions of porcine monocyte-derived DCs (moDC) with a virulent (22653/14), a low virulent (NH/P68) and an avirulent (BA71V) ASFV strain. At a high multiplicity of infection (MOI = 1), all three strains infected immature moDC. Maturation of moDC, with IFN-α/TNF-α, increased susceptibility to infection with 22653/14 and other virulent strains, but reduced susceptibility to NH/P68 and BA71V. The reduced moDC susceptibility to BA71V/NH/P68 was IFN-α mediated, whereas increased susceptibility to 22653/14 was induced by TNF-α. Using an MOI of 0.01, we observed that BA71V replicated less efficiently in moDC compared to the other isolates and we detected increased replication of NH/P68 compared to 22653/14. We observed that BA71V and NH/P68, but not 22653/14, downregulated expression of MHC class I on infected cells. All three strains decreased CD16 expression on moDC, whereas ASFV infection resulted in CD80/86 down-regulation and MHC class II DR up-regulation on mature moDC. None of the tested strains induced a strong cytokine response to ASFV and only modest IL-1α was released after BA71V infection. Overall our results revealed differences between strains and suggest that ASFV has evolved mechanisms to replicate covertly in inflammatory DC, which likely impairs the induction of an effective immune response.