Evidence of Circulation of Several HAV Genetic Variants and Emergence of Potential Antigenic Variants in an Endemo-Epidemic Country before Vaccine Introduction

Figures

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  Figure 1. Phylogenetic trees comparing nucleotide sequences of HAV strains isolated in Tunisia during 2013–2018 with the reference sequences. The tree was performed using the maximum likelihood method and the kimura-2 parameter model. Topology was supported by 1000 bootstrap replicates. The sequences reported in this study were identified by the accession numbers. The sequences of isolates reported in this study are indicated by their accession number followed by the country code and the year of isolation. Reference HAV strains are shown in italic. Clusters are designed (a–e). The same color is assigned to the same sequences grouped in the same cluster. (A). Phylogenetic tree comparing 884nt of the VP1 region of 52 strains from this study with 12 reference HAV strains. (B). Phylogenetic tree comparing 319 nt of the VP1-2A region of 77 strains from this study with 12 reference HAV strains.

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  Figure 2. Phylogenetic tree of Tunisian HAV genotype IA isolates, constructed using the maximum likelihood method and the kimura-2 parameter model based on 168nt of the VP1-2A junction. Black squares represent isolates reported in this study (accession numbers MW117967 to MW118018 and MW222090 to MW222114). The sequences of isolates are indicated by their accession number followed by district and year of isolation. Sequences of unknown districts are indicated by their accession number followed by the country code and the year of isolation. Genetic variants are represented in different colors. Topology was supported by 1000 bootstrap replicates. Reference strain HM175 was used as an “outgroup”.

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  Figure 3. Phylogenetic tree based on the analysis of the whole VP1 region belonging to genotype IA, constructed using the maximum likelihood method and the kimura-2 parameter model. The tree includes HAV Tunisian 45 sequences reported in this study and 57 previously published sequences from Tunisia and 118 from other countries. Sequences from 21 different countries (Italy, France, Argentina, Chile, Uruguay, Spain, Venezuela, Brazil, USA, Mexico, China, Russia, Japan, South Korea, Mongolia, Hungary, Thailand, Vietnam, Indonesia, Singapore and Yugoslavia) were retrieved from GenBank. The sequences of isolates are indicated by their accession number and the country code. Black triangles represent Tunisian isolates. Sequences from others countries grouped with the Tunisian isolates are represented in different colors (sequences from France in red, from Spain in blue and from Yugoslavia in green). Topology was supported by 1000 bootstrap replicates. Bootstrap values lower than 50 were not indicated. Reference strain CF53 was used as an “outgroup”.

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  Figure 4. Amino acid replacements localization compared to reference strain and position within the experimentally B/Tcell epitopes regions [12,37,38,40,41,45]. Replacements located in epitope region are indicated in red. Replacements located close to epitope region are indicated in blue.

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  Figure 5. Effect of the deleted region found in the Tunisian variant on the three-dimensional structure of HAV-VP1 protein. (a) HAV capsid protein (PDB-ID: 4QPI). Gray protein corresponds to VP1, blue to VP2 and purple to VP3. (b) Top view Figure 1 protein (PDB-ID: 4QPI-A). (c) The three-dimensional predicted structure of the VP1 protein of the Tunisian variant isolated in 2018 (accession number: MW118004). The homology model was obtained using modeler and as a template HAV-VP1 protein (PDB: 4QPI-A). Circled region indicates the destroyed helix (colored in red) resulted from the deletion of 6 amino acids in the VP1 protein region.