Summary
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.
...Italian regions have experienced a significant trend to promote the private sector (e.g. in most northern regions, including Lombardy, the region most affected by the pandemic) 17,18,19. ...while ...managing the emergency, hospital care should have been assisted more by local assistance. ...there have been severe cuts to the health system funding and lack of replacement of health workers 20. Because of these political decisions, the Italian health system was unprepared to face a pandemic. ...the most economically fragile people (caregivers, housekeepers, precarious workers and all those who do not have adequate socio-economic protection) are those that will most likely have serious repercussions in terms of health implications even in the long run 25, 29.
Bovine viral diarrhea viruses (BVDV) are members of the Pestivirus genus within the family Flaviviridae. Based on antigenic and nucleotide differences, BVDV are classified into two recognized ...species, BVDV-1 and BVDV-2. More recently, a new putative pestivirus species, tentatively called “HoBi-like”, has been associated with bovine viral diarrhea. HoBi-like viruses were first identified in fetal bovine serum (FBS) imported from Brazil. Subsequently, a number of HoBi-like viruses have been detected as contaminants in FBS or cell culture and in live ruminants. To further investigate the possible pestivirus contamination in commercially available FBS batches, 26 batches of FBS with various countries of origin, were tested in this study for the presence of bovine pestiviruses. All the 26 batches were positive by RT-PCR for at least one species of bovine pestiviruses. HoBi-like viruses were detected in 15 batches. Analysis of the 5′UTR and Npro sequences of 15 newly identified HoBi-like viruses combined with analysis of additional sequences from GenBank, identified 4 genetic groups tentatively named 3a–3d. The current study confirmed the presence of the emerging HoBi-like viruses in FBS products labeled with different geographic origins. This finding has obvious implications for the safety of biological products, such cell lines and vaccines.
Mesenchymal stromal cells (MSCs) are multipotent somatic cells that can differentiate into a variety of mature cell types. Over recent years, their biological in vitro and in vivo properties have ...elicited great expectations in the field of regenerative medicine, immunotherapy and tumour treatment. An increasing number of experimental observations suggest that their biological effects are probably related to a paracrine mechanism via the release of trophic factors and cytokines as well as through the production of membrane vesicles (MVs). These are nanometric membrane-bound structures, comprising shedding vesicles (SV) and exosomes (Ex), that enclose and transfer signalling molecules to target cells. We hypothesized that MVs may be implicated in the biological effects of MSCs from horse adipose tissue (E-AdMSCs), a type of MSC that has been extensively studied in recent years for its remarkable efficacy in tissue regeneration. By means of electron microscopy, we ascertained, for the first time, that equine adipose-derived MSCs constitutively produce MVs (E-Ad-MSCs). The analysis of MVs separated by ultracentrifugation allowed us to describe their general morphological features. Through the examination of cell monolayers by TEM, additionally, we distinguished the different pathways of SV and Ex formation, demonstrating that both fractions are produced by E-AdMSC. The accurate description of MV heterogeneous morphological characteristics led us to emphasize the possible implications of the relationship between different morphologies versus different functions. The data presented in this paper has an additional value, as they can be noteworthy for horses as well as for other mammalian species, including humans.
Summary Two European laboratories independently detected atypical bovine pestiviral nucleic acids in three commercial batches of foetal bovine serum (FBS) that was claimed by the producers to be of ...Australian origin. Additional batches of FBS were obtained directly from Australia to exclude possible contamination of the Australian FBS with that of South American origin during manufacturing/packaging in European countries. RT‐PCR amplification of partial 5′untranslated region and the complete Npro gene yielded a specific band with expected size, which was confirmed by DNA sequencing. Bayesian analysis of sequence data demonstrated a closer phylogenetic relation of the newly detected atypical bovine pestiviruses to those of South American origin, which were related to the recognized bovine pestivirus species, bovine viral diarrhoea virus. Taken together, the results indicated the presence of atypical bovine pestiviruses in the Australian FBS, and most likely in Australian Continent.
In order to investigate the genetic heterogeneity of small ruminant lentivirus (SRLV) isolates in Italy, 55 clinical samples collected between 1998 and 2010 were analysed. The phylogenetic study was ...based on analysis of gag–pol sequences. Our findings revealed that the SRLVs belonged to the subtype A9 (n = 3, sheep), B1 (n = 5, goat), B2 (n = 3, sheep) and E2 (n = 5, goat). Interestingly, 39 isolates from both sheep and goat, significantly differed from all the other SRLVs previously described and formed two separate clusters within genotypes A and B tentatively named A11 (n = 27, goat and sheep) and B3 (n = 12, goat and sheep), which have never been shown before. These results revealed a marked diversity among Italian field SRLV strains which might reflect the absence of any systematic control measures.
Plasma membrane lipid microdomains have been considered as a sort of 'closed chamber', where several subcellular activities, including CD95/Fas-mediated proapoptotic signaling, take place. In this ...work we detected GD3 and GM3 gangliosides in isolated mitochondria from lymphoblastoid CEM cells. Moreover, we demonstrated the presence of microdomains in mitochondria by immunogold transmission electron microscopy. We also showed that GD3, the voltage-dependent anion channel-1 (VDAC-1) and the fission protein hFis1 are structural components of a multimolecular signaling complex, in which Bcl-2 family proteins (t-Bid and Bax) are recruited. The disruption of lipid microdomains in isolated mitochondria by methyl-beta-cyclodextrin prevented mitochondria depolarization induced by GD3 or t-Bid. Thus, mitochondrion appears as a subcompartmentalized organelle, in which microdomains may act as controllers of their apoptogenic programs, including fission-associated morphogenetic changes, megapore formation and function. These results disclose a new scenario in which mitochondria-associated lipid microdomains can act as regulators and catalysts of cell fate.
Synthetic peptides were generated, corresponding to SU5 domain of envelope glycoprotein of Italian SRLV isolates It-561 and It-Pi1, belonging respectively to MVV- and CAEV-like genotypes. The ...peptides, encompassing an N-terminal variable and a C-terminal conserved antibody-binding site, were used in an ELISA assay to analyse the sera of two groups of sheep experimentally infected with these isolates. The kinetics and specificity of the humoral response to the homologous and heterologous antigen and the affinity maturation of the sera were evaluated. Seroconversion occurred between week 3 and 8. The response to SU5 antigen was mostly type-specific. The few broadly reacting sera may reflect the production of antibodies directed to the SU5 constant antibody-binding site. All sera underwent with time avidity maturation, resulting in the appearance of high affinity antibodies. This study suggests constant monitoring of the circulating viral variants to develop a panel of diagnostic peptides representative of local genotypes.
Summary
Currently, the genus Pestivirus comprises four approved species, namely bovine viral diarrhoea viruses 1 and 2 (BVDV‐1, BVDV‐2), classical swine fever virus and border disease virus (BDV). ...Recently, three major genotypes have been identified within the species BDV and termed as subgroups BDV‐1, BDV‐2 and BDV‐3. Here, an isolate from animals in a herd showing BD‐like syndromes, which occurred in central Italy was analysed. A reverse transcriptase polymerase chain reaction was performed using primers that specifically amplify a fragment of the 5′‐non‐coding region (5′‐NCR) from BDV. Both the 5′‐NCR fragment and the entire Npro gene were sequenced and used for genetic typing. The 5′‐NCR sequence revealed that the newly isolated Pestivirus could be allocated to the BDV species. Interestingly, the Npro sequence of this virus isolate significantly differed from all the ovine pestiviruses previously described, providing evidence for the presence of an additional subgroup within the species BDV.
The effect of subchronic intracerebroventricular injection of the human immunodeficiency virus type 1 (HIV-1) recombinant protein gp120 (100
ng, given daily for up to seven consecutive days) on ...interleukin-1
β expression was studied by immunohistochemistry in the brain of adult rats. In comparison to control, bovine serum albumin (300
ng, given intracerebroventricularly for up to seven days) -treated animals (
n=6), interleukin-1
β immunoreactivity increased in the brain cortex and hippocampus of rats (
n=6) receiving a single injection of the viral protein 24
h before analysis with more substantial increases being observed in these regions of the brain (
n=6) after seven days treatment. Double-labelling immunofluorescence experiments support a neuronal and, possibly, a microglial cell origin for gp120-enhanced interleukin-1
β expression. Transmission electron microscopy analysis of brain tissue sections revealed that combination treatments (given intracerebroventricularly daily for seven days) with gp120 (100
ng) and interleukin-1 receptor antagonist (80
ng) or with the interleukin converting enzyme inhibitor II (100
pmol), but not with leupeptin (100
pmol), prevented apoptotic death of rat (
n=6/group) brain cortical cells typically elicited by the viral protein. These data demonstrate that gp120 enhances interleukin-1
β expression in the brain and this may be involved in the mechanism underlying apoptosis induced by gp120 in the brain cortex of rat. Further support to this hypothesis comes from the evidence that intracerebroventricular injection of murine recombinant interleukin-1
β (200
U, given daily for seven consecutive days) produces DNA fragmentation in the brain cortex of rat (
n=6). Interestingly, the latter treatment enhanced nerve growth factor level in the hippocampus but not in the cerebral cortex and this coincides with a similar effect recently reported in identical brain areas of rats treated likewise with gp120.
In conclusion, the present data demonstrate that treatment with gp120 enhances interleukin-1
β expression and this participates in the mechanism of apoptotic cell death in the brain cortex of rat. By contrast, in the hippocampus, gp120-enhanced interleukin-1
β expression elevates nerve growth factor that may prevent or delay apoptosis in this plastic region of the rat brain.
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