ABSTRACT
Chronic HBV + HDV infection is associated with greater risk of liver fibrosis, earlier hepatic decompensation, and liver cirrhosis hepatocellular carcinoma compared to HBV mono-infection. However, to-date no direct anti-HDV drugs are available in clinical practice. Here, we identified conserved and variable regions in HBsAg and HDAg domains in HBV + HDV infection, a critical finding for the design of innovative therapeutic agents. The extent of amino-acid variability was measured by Shannon-Entropy (Sn) in HBsAg genotype-d sequences from 31 HBV + HDV infected and 62 HBV mono-infected patients (comparable for demographics and virological-parameters), and in 47 HDAg genotype-1 sequences. Positions with Sn = 0 were defined as conserved. The percentage of conserved HBsAg-positions was significantly higher in HBV + HDV infection than HBV mono-infection (p = 0.001). Results were confirmed after stratification for HBeAg-status and patients' age. A Sn = 0 at specific positions in the C-terminus HBsAg were correlated with higher HDV-RNA, suggesting that conservation of these positions can preserve HDV-fitness. Conversely, HDAg was characterized by a lower percentage of conserved-residues than HBsAg (p < 0.001), indicating higher functional plasticity. Furthermore, specific HDAg-mutations were significantly correlated with higher HDV-RNA, suggesting a role in conferring HDV replicative-advantage. Among HDAg-domains, only the virus-assembly signal exhibited a high genetic conservation (75% of conserved-residues). In conclusion, HDV can constrain HBsAg genetic evolution to preserve its fitness. The identification of conserved regions in HDAg poses the basis for designing innovative targets against HDV-infection.
Subject(s)
Evolution, Molecular , Hepatitis B Surface Antigens/genetics , Hepatitis B virus/genetics , Hepatitis B/virology , Hepatitis Delta Virus/physiology , Microbial Interactions , Adult , Antiviral Agents/pharmacology , Coinfection , Female , Genetic Variation , Genotype , Hepatitis B/diagnosis , Hepatitis B Surface Antigens/chemistry , Hepatitis B virus/drug effects , Humans , Male , Middle Aged , Models, Molecular , Mutation , Phylogeny , Protein Conformation , RNA, Viral , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
Clinically relevant yeasts are conventionally identified by a combination of phenotypic tests, which occasionally provide ambiguous results for atypical isolates or uncommon species. In this study, we evaluate a direct polymerase chain reaction-sequencing method, which exploits sequence divergence in the hypervariable D2 region of the large subunit of the 25-28S ribosomal RNA (rRNA) gene for identification of facultative pathogenic asco- and basidiomycota. A panel of 53 yeasts, including 40 clinical isolates and 13 reference strains representative of some clinically relevant taxa, was investigated by combining standard phenotypic tests with commercial identification systems (RapID, API 20C AUX), and results were compared with the taxonomic allocations inferred by D2 sequence analysis. Species-level resolution was achieved for almost all (52/53) strains by combining internet-based D2 sequence homology (BLAST and FASTA) searches in free-access synchronised databases with phylogenetic analysis. The phylogenetic information carried by the short D2 sequence substantiates a pattern of molecular evolution, which is similar to that inferred from analysis of the larger D1/D2 region, and consistent with previously published 25-28S rRNA phylogenetic architectures of facultative pathogenic yeast, including recently identified species. Inconsistency between conventional and molecular identification results was observed for 11/53 strains, likely on account of the ambiguous interpretation of phenotypic tests.
