One health surveillance strategy for coronaviruses in Italian wildlife.

The recent reinforcement of CoV surveillance in animals fuelled by the COVID-19 pandemic provided increasing evidence that mammals other than bats might hide further diversity and play critical roles in human infectious diseases. This work describes the results of a two-year survey carried out in Italy with the double objective of uncovering CoV diversity associated with wildlife and of excluding the establishment of a reservoir for SARS-CoV-2 in particularly susceptible or exposed species. The survey targeted hosts from five different orders and was harmonised across the country in terms of sample size, target tissues, and molecular test. Results showed the circulation of 8 CoV species in 13 hosts out of the 42 screened. Coronaviruses were either typical of the host species/genus or normally associated with their domestic counterpart. Two novel viruses likely belonging to a novel CoV genus were found in mustelids. All samples were negative for SARS-CoV-2, with minimum detectable prevalence ranging between 0.49% and 4.78% in the 13 species reaching our threshold sample size of 59 individuals. Considering that within-species transmission in white-tailed deer resulted in raising the prevalence from 5% to 81% within a few months, this result would exclude a sustained cycle after spillback in the tested species.

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 PCV2a was the first genotype detected with the highest frequency from the late 1990s to 2000, which was then superseded by PCV2b (first genotype shift).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.

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.

First Genomic Evidence of Dual African Swine Fever Virus Infection: Case Report from Recent and Historical Outbreaks in Sardinia.

African swine fever virus (ASFV) is one of the pathogens of highest concern worldwide. Despite different virus lineages co-circulating in several areas, dual infections in the same animal have been rarely observed, suggesting that ASF superinfections are infrequent events. Here we present the first genome-wide detection and analysis of two intragenotype dual ASFV infections. The dual infections have been detected in a hunted wild boar and in a pig carcass, both infected by ASFV genotype I in Sardinia in 1984 and 2018, respectively. We characterize the genetic differences between the two sequences, their intra-host frequency, and their phylogenetic relationship among fully sequenced ASFV strains from Sardinia. Both dual infections involve pairs of closely related but different viruses that were circulating in Sardinia in the same period. The results imply that dual ASFV infections or similar ASFV strains are more common than expected, especially in ASF endemic areas, albeit difficult to detect.

Surveillance and control of African Swine Fever in free-ranging pigs in Sardinia.

African swine fever (ASF) is a notifiable infectious disease, caused by the ASF virus (ASFV), which is a DNA virus belonging to the family Asfarviridae, genus Asfivirus. This disease has gained importance in the last decade after its spread in several countries in Eastern and Central Europe, and more recently, in China. Despite the efforts made to eradicate it, ASF is still present on the Mediterranean island of Sardinia (Italy) and has been since 1978. ASF risk factors on the island have been analysed in previous studies; the role of free-ranging pigs in virus persistence has been suggested, but has not been fully elucidated. The most recent eradication plan provides more stringent measures to combat free-ranging pigs and any kind of illegality in the pig sector. From December 2017 to June 2018, a total of 29 depopulation actions were performed in 13 municipalities in central Sardinia, during which 2,281 free-ranging pigs were culled and more than 50% of them were tested for ASFV and antibody presence (1,218 and 1,416, respectively). A total of 651 pigs were seropositive, with a mean seroprevalence of 53.4% (CI 95% = 50.6-56.3), and 38 were ASFV positive (virus prevalence = 2.6%; CI 95% = 2.1-3.0). To the best of our knowledge, the present study is the first to provide a complete evaluation of this millennial system of pig farming and ASFV prevalence in free-ranging pigs. Furthermore, it has emphasised the necessity of combining the maintenance of an epidemiological surveillance program with continuous education of farmers and other people involved in pig husbandry, based on cultural and economic aspects.