In vitro phenotypic characterisation of two genotype I African swine fever viruses with genomic deletion isolated from Sardinian wild boars.

African swine fever virus (ASFV) causes a devastating disease affecting domestic and wild pigs. ASF was first introduced in Sardinia in 1978 and until 2019 only genotype I isolates were identified. A remarkable genetic stability of Sardinian ASFV isolates was described, nevertheless in 2019 two wild boar isolates with a sustained genomic deletion (4342 base pairs) were identified (7303WB/19, 7212WB/19). In this study, we therefore performed in vitro experiments with monocyte-derived macrophages (moMФ) to unravel the phenotypic characteristics of these deleted viruses. Both 7303WB/19 and 7212WB/19 presented a lower growth kinetic in moMФ compared to virulent Sardinian 26544/OG10, using either a high (1) or a low (0.01) multiplicity of infection (MOI). In addition, flow cytometric analysis showed that both 7303WB/19 and 7212WB/19 presented lower intracellular levels of both early and late ASFV proteins. We subsequently investigated whether deleted virus variants were previously circulating in wild boars in Sardinia. In the four years preceding the last genotype I isolation (February 2015-January 2019), other eight wild boar isolates were collected, all belonging to p72 genotype I, B602L subgroup X, but none of them presented a sustained genomic deletion. Overall, we observed the deleted virus isolates in Sardinia only in 2019, at the end of a strong eradication campaign, and our data suggest that it might possess an attenuated phenotype in vivo. A better understanding of ASFV evolution in endemic territories might contribute to development of effective control measures against ASF.

Evidence of Porcine Circovirus Type 2 (PCV2) Genetic Shift from PCV2b to PCV2d Genotype in Sardinia, Italy.

Porcine Circovirus type 2 (PCV2) is the etiological agent of a disease syndrome named Porcine Circovirus disease (PCVD), representing an important threat for the pig industry. The increasing international trade of live animals and the development of intensive pig farming seem to have sustained the spreading of PCVD on a global scale. Recent classification criteria allowed the identification of nine different PCV2 genotypes (PCV2a-i). PCV2a was the first genotype detected with the highest frequency from the late 1990s to 2000, which was then superseded by PCV2b (first genotype shift). An ongoing genotype shift is now determining increasing prevalence rates of PCV2d, in replacement of PCV2b. In Italy, a complete genotype replacement was not evidenced yet. The present study was carried out on 369 samples originating from domestic pigs, free-ranging pigs, and wild boars collected in Sardinia between 2020 and 2022, with the aim to update the last survey performed on samples collected during 2009-2013. Fifty-seven complete ORF2 sequences were obtained, and the phylogenetic and network analyses evidenced that 56 out of 57 strains belong to the PCV2d genotype and only one strain to PCV2b, thus showing the occurrence of a genotype shift from PCV2b to PCV2d in Sardinia.

The Synergic Role of Emerging and Endemic Swine Virus in the Porcine Respiratory Disease Complex: Pathological and Biomolecular Analysis.

Porcine respiratory disease complex (PRDC) represents a significant threat to the swine industry, causing economic losses in pigs worldwide. Recently, beyond the endemic viruses PRRSV and PCV2, emerging viruses such as TTSuV, PCV3, and PPV2, have been associated with PRDC, but their role remains unclear. This study investigates the presence of PCV2 and PRRSV and emerging viruses (PCV3, TTSuV, and PPV2) in the lungs of swine belonging to different age groups by histopathology and real-time PCR. The prevalent lung lesion was interstitial pneumonia with increased severity in post-weaning pigs. PRRSV was detected in 33% of piglets' lungs and in 20% of adults and post-weaning pigs with high Ct, while PCV2 was found in 100% of adult pigs, 33% of post-weaning pigs, and 22% of piglets, with low Ct in post-weaning pigs. PCV3 was present in all categories and coexisted with other viruses. TTSuV was detected in all swine in combination with other viruses, possibly influencing the disease dynamics, while PPV2 was detected in 100% of adults' and 90% of piglets' lungs. The detection of TTSuV, PCV3, and PPV2 in affected pigs prioritizes the need for comprehensive approaches in implementing appropriate control measures and minimizing economic losses associated with PRDC.

Development of molecular and antigenic-based rapid tests for the identification of African swine fever virus in different tissues.

African swine fever (ASF) is a severe haemorrhagic infectious disease affecting suids, thus representing a great economic concern. Considering the importance of the early diagnosis, rapid point of care testing (POCT) for ASF is highly demanded. In this work, we developed two strategies for the rapid onsite diagnosis of ASF, based on Lateral Flow Immunoassay (LFIA) and Recombinase Polymerase Amplification (RPA) techniques. The LFIA was a sandwich-type immunoassay exploiting a monoclonal antibody directed towards the p30 protein of the virus (Mab). The Mab was anchored onto the LFIA membrane to capture the ASFV and was also labelled with gold nanoparticles for staining the antibody-p30 complex. However, the use of the same antibody for capturing and as detector ligand showed a significant competitive effect for antigen binding, so required an experimental design to minimize reciprocal interference and maximize the response. The RPA assay, employing primers to the capsid protein p72 gene and an exonuclease III probe, was performed at 39 °C. The limit of detection of the method was assessed using a plasmid encoding the target gene and resulted in 5 copy/µL. The new LFIA and RPA were applied for ASFV detection in the animal tissues usually analysed by conventional assays (i.e., real-time PCR), such as kidney, spleen, and lymph nodes. A simple and universal virus extraction protocol was applied for sample preparation, followed by DNA extraction and purification for the RPA. The LFIA only required the addition of 3% H2O2 to limit matrix interference and prevent false positive results. The two rapid methods (25 min and 15 min were needed to complete the analysis for RPA and LFIA, respectively) showed high diagnostic specificity (100%) and sensitivity (93% and 87% for LFIA and RPA, respectively) for samples with high viral load (Ct < 27). False negative results were observed for samples with low viral load (Ct > 28) and/or also containing specific antibodies to ASFV, which decreased antigen availability and were indicative of a chronic, poorly transmissible infection. The simple and rapid sample preparation and the diagnostic performance of the LFIA suggested its large practical applicability for POC diagnosis of ASF.

Origin, Genetic Variation and Molecular Epidemiology of SARS-CoV-2 Strains Circulating in Sardinia (Italy) during the First and Second COVID-19 Epidemic Waves.

Understanding how geography and human mobility shape the patterns and spread of infectious diseases such as COVID-19 is key to control future epidemics. An interesting example is provided by the second wave of the COVID-19 epidemic in Europe, which was facilitated by the intense movement of tourists around the Mediterranean coast in summer 2020. The Italian island of Sardinia is a major tourist destination and is widely believed to be the origin of the second Italian wave. In this study, we characterize the genetic variation among SARS-CoV-2 strains circulating in northern Sardinia during the first and second Italian waves using both Illumina and Oxford Nanopore Technologies Next Generation Sequencing methods. Most viruses were placed into a single clade, implying that despite substantial virus inflow, most outbreaks did not spread widely. The second epidemic wave on the island was actually driven by local transmission of a single B.1.177 subclade. Phylogeographic analyses further suggest that those viral strains circulating on the island were not a relevant source for the second epidemic wave in Italy. This result, however, does not rule out the possibility of intense mixing and transmission of the virus among tourists as a major contributor to the second Italian wave.

The Long-Jumping of African Swine Fever: First Genotype II Notified in Sardinia, Italy.

African swine fever (ASF) is a devastating infectious disease of domestic pigs and wild boar that is spreading quickly around the world and causing huge economic losses. Although the development of effective vaccines is currently being attempted by several labs, the absence of globally recognized licensed vaccines makes disease prevention and early detection even more crucial. ASF has spread across many countries in Europe and about two years ago affected the Italian susceptible population. In Italy, the first case of ASF genotype II in wild boar dates back to January 2022, while the first outbreak in a domestic pig farm was notified in August 2023. Currently, four clusters of infection are still ongoing in northern (Piedmont-Liguria and Lombardy), central (Lazio), and southern Italy (Calabria and Campania). In early September 2023, the first case of ASFV genotype II was detected in a domestic pig farm in Sardinia, historically affected by genotype I and in the final stage of eradication. Genomic characterization of p72, p54, and I73R/I329L genome regions revealed 100% similarity to those obtained from isolates that have been circulating in mainland Italy since January 2022 and also with international strains. The outbreak was detected and confirmed due to the passive surveillance plan on domestic pig farms put in place to provide evidence on genotype I's absence. Epidemiological investigations suggest 24 August as the most probable time of ASFV genotype II's arrival in Sardinia, likely due to human activities.

A Naturally Occurring Microhomology-Mediated Deletion of Three Genes in African Swine Fever Virus Isolated from Two Sardinian Wild Boars.

African swine fever virus (ASFV) is the etiological agent of a lethal disease of domestic pigs and wild boars. ASF threatens the pig industry worldwide due to the lack of a licensed vaccine or treatment. The disease has been endemic for more than 40 years in Sardinia (Italy), but an intense campaign pushed it close to eradication; virus circulation was last detected in wild boars in 2019. In this study, we present a genomic analysis of two ASFV strains isolated in Sardinia from two wild boars during the 2019 hunting season. Both isolates presented a deletion of 4342 base pairs near the 5' end of the genome, encompassing the genes MGF 360-6L, X69R, and MGF 300-1L. The phylogenetic evidence suggests that the deletion recently originated within the Sardinia ecosystem and that it is most likely the result of a non-allelic homologous recombination driven by a microhomology present in most Sardinian ASFV genomes. These results represent a striking example of a genomic feature promoting the rapid evolution of structural variations and plasticity in the ASFV genome. They also raise interesting questions about the functions of the deleted genes and the potential link between the evolutionary timing of the deletion appearance and the eradication campaign.

A Review on Pathological and Diagnostic Aspects of Emerging Viruses-Senecavirus A, Torque teno sus virus and Linda Virus-In Swine.

Swine production represents a significant component in agricultural economies as it occupies over 30% of global meat demand. Infectious diseases could constrain the swine health and productivity of the global swine industry. In particular, emerging swine viral diseases are omnipresent in swine populations, but the limited knowledge of the pathogenesis and the scarce information related to associated lesions restrict the development of data-based control strategies aimed to reduce the potentially great impact on the swine industry. In this paper, we reviewed and summarized the main pathological findings related to emerging viruses, such as Senecavirus A, Torque teno sus virus, and Linda virus, suggesting a call for further multidisciplinary studies aimed to fill this lack of knowledge and better clarify the potential role of those viral diseases in swine pathology.

A Deeper Insight into Evolutionary Patterns and Phylogenetic History of ASFV Epidemics in Sardinia (Italy) through Extensive Genomic Sequencing.

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.

Genetic Characterization of Porcine Circovirus 3 Strains Circulating in Sardinian Pigs and Wild Boars.

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.

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

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.

Interaction of historical and modern Sardinian African swine fever viruses with porcine and wild-boar monocytes and monocyte-derived macrophages.

African 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 ASF virus (ASFV) strains circulating in Sardinia from 1990 onwards, and modern Sardinian strains (isolated after 1990) might have acquired some selective advantage compared to historical ones (isolated before 1990). Here, we analysed the host cell responses of wild boars and domestic pigs upon infection with virus variants. Higher intracellular levels of the late protein p72 were detected after infection with the modern strain 22653/14 compared to the historical strain Nu81.2, although both isolates grew at the same rate in both monocytes and monocyte-derived macrophages. Higher cytokine levels in the supernatants of ASFV-infected pig monocytes compared to pig macrophages and wild-boar cells were detected, with no differences between isolates.