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Over the last few decades, several pestiviruses have been discovered in ruminants, pigs, and, more recently, in non-ungulate hosts. Consequently, the nomenclature and taxonomy of pestiviruses have been updated. The Tunisian sheep-like pestivirus (TSV, Pestivirus N ) is an additional ovine pestivirus genetically closely related to classical swine fever virus (CSFV). In this study, during a survey of pestivirus infections in ovine farms in the Lombardy region of Northern Italy, we identified and isolated a pestivirus strain from a sheep that was found to belong to Pestivirus N species based on its genomic nucleotide identity. The sheep itself and its lamb were found to be persistently infected. We performed molecular characterization and phylogenetic analysis of three viral genomic regions (a fragment of 5'-UTR, partial N pro , and the whole E2 region). In conclusion, these results confirmed circulating TSV in Northern Italy after notification in Sicily, Italy, and France. Correlation with Italian, Tunisian, and French strains showed that detection might have resulted from the trading of live animals between countries, which supports the need for health control measures.

This study shows the origin and the pathogenic role of a novel ovine pestivirus (OVPV) isolated in 2017 in Italy, as a pathogenic agent causing severe abortions after infection in pregnant ewes and high capacity for virus trans-placental transmission as well as the birth of lambs suffering OVPV-persistent infection. The OVPV infection induced early antibody response detected by the specific ELISA against classical swine fever virus (CSFV), another important virus affecting swine. The neutralizing antibody response were similar against CSFV strains from genotype 2 and the OVPV. These viruses showed high identity in the B/C domain of the E2-glycoprotein. Close molecular diagnostics cross-reactivity between CSFV and OVPV was found and a new OVPV molecular assay was developed. The phylodynamic analysis showed that CSFV seems to have emerged as the result of an inter-species jump of Tunisian sheep virus (TSV) from sheep to pigs. The OVPV and the CSFV share the TSV as a common ancestor, emerging around 300 years ago. This suggests that the differentiation of TSV into two dangerous new viruses for animal health (CSFV and OVPV) was likely favored by human intervention for the close housing of multiple species for intensive livestock production.

Atypical porcine pestivirus (APPV) is a newly recognized member of the Flaviviridae family. This novel porcine pestivirus was first described in 2015 in the USA, where it has been associated with congenital tremor type A-II in new-born piglets. APPV is widely distributed in domestic pigs in Europe and Asia. In this study, a virological survey was performed in Northern Italy to investigate the presence of APPV using molecular methods. Testing of 360 abortion samples from pig herds revealed two APPV strains from distinct provinces in the Lombardy region and testing of 430 wild boar blood samples revealed three strains, one from Lombardy and two from Emilia Romagna. The nucleotide sequencing of a fragment of the nonstructural protein 3-coding region revealed a high similarity to the previously detected European strains (Spanish, German, and Italian) of APPV.

Pestiviruses are distributed worldwide and are responsible for a variety of economically important diseases. They are not very host-specific, and thus sheep can be infected by well-known pestiviruses like bovine viral diarrhea virus (BVDV) and border disease virus (BDV), as well as by other recently discovered pestivirus species. The aim of this study is to describe the isolation and characterization of four pestivirus strains detected in aborted lamb fetuses from a single farm in the Brescia province (Northern Italy). A total of twelve aborted fetuses were collected and examined. After necropsy, organs were tested for the presence of infectious agents known as potential causes of abortion ( Brucella spp., Listeria spp., Coxiella burnetii , Chlamydophila spp., Mycoplasma spp., Neospora caninum , and Toxoplasma gondii ), and submitted to viral identification by isolation on Madin Darby bovine kidney (MDBK) cell culture and by PCR assay for Schmallenberg virus and pan-pestivirus RT-PCR real time assay. Three viral strains (Ovine/IT/1756/2017, Ovine/IT/338710-2/2017, and Ovine/IT/338710-3/2017) were isolated in the absence of cytopathic effects (CPEs) in cell cultures and identified with RT-PCR. Another pestivirus strain (Ovine/IT/16235-2/2018) was detected by PCR, but was not successfully isolated. Complete sequence genomic data of the three isolated viruses showed that they were highly similar, differed genetically from known pestivirus species, and were closely related to classical swine fever virus (CSFV). Beyond the identification of new ovine pestiviruses, this study indicates that a systematic diagnostic approach is important to identify the presence and map the distribution of both known and emerging pestiviruses.