Genomic analysis of Sardinian 26544/OG10 isolate of African swine fever virus

Abstract Comparative genomic analysis aims to underscore genetic assortment diversification in distinct viral isolates, to identify deletions and to carry out evolutionary studies. We sequenced the first complete genome of an ASFV p72 genotype I strain isolated from domestic pigs in Sardinia (Italy) using Next-Generation Sequence (NGS) technology. The genome is 182,906 bp long, contains 164 ORFs and has a 99.20% nucleotide identity to the L60 strain. Comparison analysis against the 16 ASFV genomes available in the database showed that 136 ORFs are present in nine ASFV isolates annotated to date. The most divergent ORFs codify for uncharacterized proteins such as X69R and DP96R, which have 51.3% and 70.4% nucleotide identity to the other isolates. A comparison between the Sardinian isolate and the avirulent isolates OURT 88/3, NHV, BA71V was also carried out. Major variations were found within the multigene families (MGFs) located in the left and right genome regions

Epidemiology and genetic characterization of <i>Border Disease Virus</i> circulating in Sardinia

Border Disease Virus (BDV), a pestivirus from the Flaviviridae family, is an important pathogen of sheep and goats responsible for significant losses in farms around the world. In spite of the relevance of this pathogen there are only a few epidemiological studies on BDV infection and, as a consequence, the economic impact on small ruminant productions is probably under-estimated. The aims of this study are i) to determine the distribution of BDV in small ruminant farms in Sardinia and genetically characterize circulating strains ii) analyze the relation between seroprevalence, Somatic Cells Count (SCC) an milk yeld. ELISA was performed using “BVDV/MD/BDV p80 Protein Antibody Test Kit” (IDEXX) on serum of bulk tank milk (BTM) samples collected from Sardinian sheep flocks and goat herds between spring 2014 and 2015. The number of sampled farms corresponded to 8.5% of all registered farms in Sardinia. RNA was isolated using Qiamp Viral RNA mini kit from the cellular fraction of each ELISA positive BTV sample and amplified by rt-PCR using complementary primers to a highly conserved region in the untranslated regions (UTRs) of the viral genome. The amplicons were sequenced for phylogenetic analysis. Geographic distribution of collected specimen, seroprevalence and virological positive samples were analyzed via GIS (ESRI ARCGIS 10.3). ELISA screening shows a seroprevalence of 8.3% among goat farms and 10.5% among ovine farms. Ten from the ELISA positive samples were found rt-PCR positive. The sequence analysis indicates that all the amplified samples match with BDV genomes and the phylogenetic analysis revealed that all the viruses clustered in the same group classified as BDV-7. BDV-7 is the only group isolated in Sardinia so far

Phylogenetic analysis of porcine circovirus type 2 in Sardinia, Italy, shows genotype 2d circulation among domestic pigs and wild boars

Abstract Porcine circovirus type 2 (PCV2) is associated with multi-factorial syndromes, commonly known as porcine-circovirus–associated diseases, which cause severe economic losses in the swine industry worldwide. Four genotypes (PCV2a, PCV2b, PCV2c, and PCV2d) have been identified. Lately, the prevalence of PCV2d has been increasing in many countries, thereby prefiguring a global replacement of PCV2b. Wild boars are also susceptible to PCV2 infection, with virus prevalence similar to that of domestic pigs. This work was aimed at expanding the knowledge about the molecular epidemiology of PCV2 in Italy. For this purpose, we analysed 40 complete ORF-2 sequences from PCV2 strains isolated from domestic pigs and wild boars in Sardinia (Italy) over a period of 5 years (2009–2013). Phylogenetic and Bayesian analyses were performed on three data sets compiled from DNA sequences over a large geographical area. PCV2b was found to be dominant in Sardinia, whereas no PCV2a and PCV2c were found. This study indicates the presence of genotype PCV2d-2 infecting both domestic and wild pigs, thus confirming its circulation in Italy. Sardinian sequences clustered mostly with Italian isolates and with strains from China, Belgium, Croatia, Taiwan, Korea, and Portugal. Genetic variability of PCV2 in Sardinia appears to be a result of both local viral evolution and different epidemic introduction events

Characterization of the interaction of African swine fever virus with monocytes and derived macrophage subsets.

Abstract 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

Evaluation of Haematological and Immunological Parameters of the ASFV Lv17/WB/Rie1 Strain and Its Derived Mutant Lv17/WB/Rie1/d110-11L against ASFV Challenge Infection in Domestic Pigs

African swine fever virus (ASFV) is the etiological agent of a haemorrhagic disease that threatens the global pig industry. There is an urgency to develop a safe and efficient vaccine, but the knowledge of the immune&ndash;pathogenetic mechanisms behind ASFV infection is still very limited. In this paper, we evaluated the haematological and immunological parameters of domestic pigs vaccinated with the ASFV Lv17/WB/Rie1 strain or its derived mutant Lv17/WB/Rie1/d110-11L and then challenged with virulent Armenia/07 ASFV. Circulating levels of C-reactive protein (CRP), 13 key cytokines and 11 haematological parameters were evaluated throughout the study. Lv17/WB/Rie1 triggered an inflammatory response, with increased levels of CRP and pro-inflammatory cytokines, and induced lymphopenia, thrombocytopenia and a decline in red blood cell (RBC) parameters, although this was transitory. Lv17/WB/Rie1/d110-11L triggered only transitory thrombocytopenia and a mild inflammatory reaction, with no increase in serum levels of pro-inflammatory cytokines, but it raised IL-1Ra levels. Both strains counteracted several adverse reactions elicited by virulent challenge, like thrombocytopenia, a decline in RBC parameters, and inflammation. Within this paper, we provided a deep portrayal of the impact of diverse ASFV strains on the domestic pig&rsquo;s immune system. A better understanding of these immune&ndash;pathological mechanisms would help to design suitable vaccines against this disease

Goat milk extracellular vesicles: immuno-modulation effects on porcine monocyte-derived macrophages in vitro

IntroductionExtracellular vesicles (EVs) are nanometric-membrane-bound sub-cellular structures, which can be recovered from milk. Milk EVs have drawn increasing interest due to their potential biomedical applications, therefore it is important to investigate their impact on key immune cells, such as macrophages.MethodsIn this work, the immunomodulatory effects of goat milk EVs on untreated (moMФ) and classically activated (moM1) porcine monocyte-derived macrophages were investigated using flow cytometry, ELISA, and gene expression assays.ResultsThese particles were efficiently internalized by macrophages and high doses (60 mg protein weight) triggered the upregulation of MHC I and MHC II DR on moMФ, but not on moM1. In moMФ, exposure to low doses (0.6 mg) of mEVs enhanced the gene expression of IL10, EBI3, and IFNB, whereas high doses up-regulated several pro-inflammatory cytokines. These nanosized structures slightly modulated cytokine gene expression on moM1. Accordingly, the cytokine (protein) contents in culture supernatants of moMФ were mildly affected by exposure to low doses of mEVs, whereas high doses promoted the increased release of TNF, IL-8, IL-1a, IL-1b, IL-1Ra, IL-6, IL-10, and IL-12. The cytokines content in moM1 supernatants was not critically affected.DiscussionOverall, our data support a clinical application of these molecules: they polarized macrophages toward an M1-like phenotype, but this activation seemed to be controlled, to prevent potentially pathological over-reaction to stressors

Interaction of historical and modern Sardinian African swine feverviruses with porcine and wild?boar monocytes and monocyte?derivedmacrophages

AbstractAfrican swine fever (ASF) is a contagious viral disease of wild and domestic pigs that is present in many parts of Africa,Asia and Europe, including Sardinia (Italy). Deletions in the EP402R and B602L genes have been found in almost all ASFvirus (ASFV) strains circulating in Sardinia from 1990 onwards, and modern Sardinian strains (isolated after 1990) mighthave acquired some selective advantage compared to historical ones (isolated before 1990). Here, we analysed the host cellresponses of wild boars and domestic pigs upon infection with virus variants. Higher intracellular levels of the late proteinp72 were detected after infection with the modern strain 22653/14 compared to the historical strain Nu81.2, although bothisolates grew at the same rate in both monocytes and monocyte-derived macrophages. Higher cytokine levels in the supernatantsof ASFV-infected pig monocytes compared to pig macrophages and wild-boar cells were detected, with no differencesbetween isolates

Small ruminant lentivirus genotype E is widespread in Sarda goat

8 p., 3 figures, 2 tables and bibliographyThe highly divergent SRLV genotype E has recently been characterized in Italy as a low pathogenic caprine lentivirus in the Roccaverano breed. The availability of a genotype specific diagnostic test based on a comparative assay, using a combination of genotype specific recombinant antigens allows a wide serosurvey in other goat populations. The island of Sardinia still has the highest small ruminant population of any Italian region and crossbreeding has been limited to goats, mainly with the Maltese breed. A serological survey was carried out on sheep flocks and goat herds, using individual sera as well as a bulk milk-adapted procedure. Genotype E was identified inmore than 50% of goat herds and none of the sheep flocks thus supporting the idea that this genotype is specifically associated with the goat species. The full-length proviral sequence of a Sardinian isolate revealed and confirmed the deletion of dUTPase subunit and the absence of both vpr gene and the 71 bp repeat of the LTR. Genetic similarity of this isolate with the prototype strain Roccaverano was not more than 84%, supporting the designation of two subtypes within genotype E. Nevertheless, in vitro properties of the Sardinian strain were different from those of the Roccaverano strain in terms of ability to infect synovial membrane and produce syncitia. Remarkable differences in the HV1 and HV2 of the env gene were recorded, with the Sardinian isolate displaying sequence motif more similar to arthritic strains. Data presented suggest diffusion of genotype E is wider than previously thought.This work has been partially funded by the Italian Ministry of Health, "Ricerca Corrente" program id IZSSA/03.Peer reviewe

Epidemiologia molecolare degli SRLV nel mediterraneo.

Trabajo presentado en el VI Workshop di Virologia Veterinaria, celebrado en Turín (Italia), los días 13 y 14 de octubre de 2016Peer reviewe

<i>Anaplasma</i> Species in Ticks Infesting Mammals of Sardinia, Italy

Ticks are hematophagous ectoparasites that are recognized for their ability to vector a wide variety of pathogens of viral, bacterial, protozoal, and helminthic nature to vertebrate hosts. Among the different diseases transmitted by ticks, also called “Tick-Borne Diseases” (TBD), many are zoonotic. Pathogens of the genus Anaplasma refer to obligate intracellular bacteria within the Rickettsiales order transmitted mainly through tick bites and considered as well-established threats to domestic animals, livestock, and humans, worldwide. In this retrospective study, 156 ticks collected from twenty goats, one marten, and one cattle from several Sardinian sites, were examined by molecular analyses to detect the presence of Anaplasma species. A total of 10 (10/156; 6.4%) ticks were shown to be Anaplasma-positive by PCR screening. After sequence analyses, A. phagocytophilum was detected in four Rhipicephalus sanguineus s.l. (3.3%) and four Rh. bursa (11%) ticks from goats, while one Rh. sanguineus s.l. (0.8%) and one Rh. bursa (2.8%) collected from the marten and cattle, respectively, exhibited 100% of identity with A. marginale strains. In this study, we provide the first description and molecular detection of A. marginale and A. phagocytophilum in ticks of the Rhiphicephalus genus in Sardinia. Considering the growing impact of tick-borne Anaplasma pathogens on human health, further studies are necessary to monitor the prevalence of these pathogens in Sardinia