From Genome Diversity to Inferred Functional Constraints: An Integrated Evolutionary Analysis of Hepatitis B Virus Genotype F

Hepatitis B virus (HBV) genotype F is one of the most genetically divergent and evolutionarily ancient HBV lineages and predominantly circulates in indigenous and admixed populations of the Americas. Here, we performed a comprehensive evolutionary and inferred functional characterization of the HBV genotype F via the largest curated dataset of complete genomes. Phylogenomic reconstruction, recombination screening, and phylogenetic network analyses were integrated with codon-based selective pressure inference, surface protein posttranslational modification profiling, mutational analysis of antigenic regions, and reverse transcriptase (RT) drug resistance assessment. The HBV-F subgenotype exhibited a well-resolved phylogenetic structure and limited intragenotypic recombination, while intergenotypic recombination contributed substantially to reticulate evolutionary signals. Selective pressure analyses revealed strong purifying selection in replication-associated domains of the polymerase, in contrast to episodic adaptive evolution in surface-exposed and regulatory proteins, particularly the X protein. N-glycosylation sites in large surface proteins are highly conserved. Some mutations in the major hydrophilic region (MHR) were significantly detected, whereas RT drug resistance mutations were rare and followed canonical lamivudine-associated pathways. Collectively, these findings highlight the balance between deep evolutionary conservation and localized adaptive flexibility in shaping the HBV genotype F and provide a genotype-specific framework for interpreting viral fitness, immune interactions, and antiviral resistance.