Braries have been prepared utilizing Illumina Nextera XT DNA Sample Preparation kit
Braries were prepared making use of Illumina Nextera XT DNA Sample Preparation kit; Illumina and sequencing was performed making use of Illumina MiSeq (two 250 bp PE). The read excellent was assessed by utilizing FastQC v0.11.two. Comprehensive genomes were generated via a reference-based strategy. The reference virus applied to receive the consensus sequences was A/barnacle goose/GermanySH/AI02167/2020 (A/H5N8) (GISAID accession number: EPI_ISL_614400). High quality reads had been aligned against a reference genome applying BWA v0.7.12 [21], processing the alignments with DY268 supplier Picard-tools v2.1.0 (http://broadinstitute.github.io/picard/; accessed on 19 October 2021) and GATK v3.5 [213]. Single nucleotide polymorphisms had been called applying LoFreq v2.1.two [24]. Viral sequences were submitted to GISAID EpiFlu Database (Supplementary Material Table S1). Sequences had been aligned employing MAFFT version 7 [25] (https://mafft.cbrc.jp/alignment/server/; accessed on 24 August 2021). Maximum likelihood phylogenetic tree in the HA gene segment was generated in IQTREE version 1.six, performing ultrafast bootstrap resampling analysis (1000 replications) [26,27]. The phylogenetic tree was visualized by using FigTree version 1.4.two [http://tree.bio.ed.ac. uk/software/figtree/; accessed on 24 August 2021]. 3. Results 3.1. Surveillance Activity 1 (Hunted Ducks) The selected wetlands on the activity had been sampled at distinctive times from 9 November 2020 to 30 January 2021 (Table 1). A total of 823 wild waterfowl belonging to 5 unique species sampled throughout the hunting activities, with Eurasian wigeons becoming one of the most several species sampled (337/823, 40.9 ), and Northern pintails the least represented (12/823, 1.5 ). Each of the molecular benefits for AIV detection obtained B-355252 Autophagy within this activity, AIV infection prevalence ( ) for each and every sampling date, hunting valley, avian species and sample material are reported in Table 1. Out of 823 hunted birds, 55 (6.7 ) tested overall optimistic for AIV in at least one particular out on the three collected samples (OS, CS and FS). Their detailed final results for HA-typing, pathotyping, NA-typing are reported in Table 3.Microorganisms 2021, 9,9 ofTable 3. Molecular pathotyping and subtyping of AIV detected in unique biological samples collected from hunted dabbling ducks (Veneto region, northeast Italy, 2020021).AIV Pos HVSS ID (yy-mm-dd) VCPa-1st (20-11-09) H-Duck ID H1_ET H2_EW H3_EW H4_EW H5_EW H6_Ga H7_Ma H8_Ma H9_Ma H10_EW H11_EW H12_EW H13_EW H14_EW H15_EW H16_EW H17_EW H18_EW H19_EW H20_EW H21_EW H22_EW H23_EW H24_EW H25_EW H26_EW H27_EW H28_EW H29_NS H30_NS H31_EW H32_EW H33_EW Sex n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. F F F F M F F F M F M M F M M F M M M F F n.a. n.a. n.a. Age n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. J J A J A J J J A J J J J A J A J J J J J n.a. n.a. n.a. AIV M Gene Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. OS Virological Final results AIV P-T n.d. n.d. n.d. n.d. HPAI n.d. n.d. n.d. HPAI HPAI HPAI n.a. n.d. n.d. HPAI HPAI n.d. n.d. HPAI n.a. n.d. HPAI n.d. HPAI HPAI n.d. n.d. n.d. n.d. n.d. HPAI n.d. HPAI AIV S-T n.d. n.d. n.d. n.d. H5N8 n.d. n.d. n.d. H5N8 H5N8 H5Nx H5Nx n.d. n.d. H5N8 H5N8 n.d. n.d. H5Nx H5Nx n.d. H5N8 n.d. H5Nx H5Nx n.d. n.d. n.d. n.d. n.d. H5N8 n.d. H5N8 AIV M Gene Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. CS Virological Outcomes AIV P-T LPAI HPAI LPAI HPAI HPAI n.d. n.d. n.d. HPAI HPAI HPAI n.d. n.d. n.a. n.d. n.d. HPAI n.d. n.a. n.d. HPAI n.d. n.d. HPAI n.d.