Phylogenetic analysis of HAV strains.

The obtained sequences, including 14 reference sequences retrieved from GenBank, were corrected using the DNASTAR Lasergene SeqMan program (DNASTAR, USA) and then aligned using MEGA6 and the Clustal W algorithm (30). In addition, three subgenotype IA sequences, two corresponding to clinical strains TunS-01-01and TunS-05-01, identified in serum samples collected in 2001 from Jammel and Monastir hospitals, and the third corresponding to environmental strain TunEU-05-01, collected from raw wastewater in the Monastir region (2001) (31), were also included in the analysis. Inter- and intragenotype nucleotide identity percentages were calculated using the DNASTAR Lasergene MegAlign program. A phylogenetic tree was constructed using the neighbor-joining algorithm (32) with calculation of distance matrices performed using the Kimura two-parameter model and a reliability test of the tree performed by bootstrap analysis (1,000 replications) with the MEGA6 program (30).