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The SARS-CoV-2 Delta variant of concern (VOC) was often associated with serious clinical course of the COVID-19 disease. Herein, we investigated the selective pressure, gene flow and evaluation on the frequencies of mutations causing amino acid substitutions in the Delta variant in three Italian regions. A total of 1500 SARS-CoV-2 Delta genomes, collected in Italy from April to October 2021 were investigated, including a subset of 596 from three Italian regions. The selective pressure and the frequency of amino acid substitutions and the prediction of their possible impact on the stability of the proteins were investigated. Delta variant dataset, in this study, identified 68 sites under positive selection: 16 in the spike (23.5%), 11 in nsp2 (16.2%) and 10 in nsp12 (14.7%) genes. Three of the positive sites in the spike were located in the receptor-binding domain (RBD). In Delta genomes from the three regions, 6 changes were identified as very common (>83.7%), 4 as common (>64.0%), 21 at low frequency (2.1%-25.0%) and 29 rare (≤2.0%). The detection of positive selection on key mutations may represent a model to identify recurrent signature mutations of the virus.

In this study, we report the first outbreak of highly pathogenic avian influenza (HPAI) A H5N8, clade 2.3.4.4b in Kosovo on 19 May 2021. The outbreak consisted of three phases: May-June 2021, September-November 2021, and January-May 2022. In total, 32 backyards and 10 commercial holdings tested positive for the virus. Interestingly, the third and last phase of the outbreak coincided with the massive H5N1 clade 2.3.4.4b epidemic in Europe. Phylogenetic analyses of 28 viral strains from Kosovo revealed that they were closely related to the H5N8 clade 2.3.4.4.b viruses that had been circulating in Albania, Bulgaria, Croatia, Hungary, and Russia in early 2021. Whole genome sequencing of the 25 and partial sequencing of three H5N8 viruses from Kosovo showed high nucleotide identity, forming a distinctive cluster and suggesting a single introduction. The results of the network analysis were in accordance with the three epidemic waves and suggested that the viral diffusion could have been caused by secondary spreads among farms and/or different introductions of the same virus from wild birds. The persistent circulation of the same virus over a one-year period highlights the potential risk of the virus becoming endemic, especially in settings with non-adequate biosecurity.