title: Novel HBsAg markers tightly correlate with occult HBV infection and strongly affect HBsAg detection.
abstract: Occult HBV infection (OBI) is a threat for the safety of blood-supply, and has been associated with the onset of HBV-related hepatocellular carcinoma and lymphomagenesis. Nevertheless, genetic markers in HBsAg (particularly in D-genotype, the most common in Europe) significantly associated with OBI in vivo are missing. Thus, the goal of this study is to define: (i) prevalence and clinical profile of OBI among blood-donors; (ii) HBsAg-mutations associated with OBI; (iii) their impact on HBsAg-detection. OBI was searched among 422,278 blood-donors screened by Nucleic-Acid-Testing. Following Taormina-OBI-definition, 26 (0.006%) OBI-patients were identified. Despite viremia <50IU/ml, HBsAg-sequences were obtained for 25/26 patients (24/25 genotype-D). OBI-associated mutations were identified by comparing OBI-HBsAg with that of 82 chronically-infected (genotype-D) patients as control. Twenty HBsAg-mutations significantly correlated for the first time with OBI. By structural analysis, they localized in the major HBV B-cell-epitope, and in HBsAg-capsid interaction region. 14/24 OBI-patients (58.8%) carried in median 3 such mutations (IQR:2.0-6.0) against 0 in chronically-infected patients. By co-variation analysis, correlations were observed for R122P+S167L (phi=0.68, P=0.01), T116N+S143L (phi=0.53, P=0.03), and Y100S+S143L (phi=0.67, p<0.001). Mutants (obtained by site-directed mutagenesis) carrying T116N, T116N+S143L, R122P, R122P+Q101R, or R122P+S167L strongly decreased HBsAg-reactivity (54.9±22.6S/CO, 31.2±12.0S/CO, 6.1±2.4S/CO, 3.0±1.0S/CO and 3.9±1.3S/CO, respectively) compared to wild-type (306.8±64.1S/CO). Even more, Y100S and Y100S+S143L supernatants show no detectable-HBsAg (experiments in quadruplicate). In conclusions, unique HBsAg-mutations in genotype-D, different than those described in genotypes B/C (rarely found in western countries), tightly correlate with OBI, and strongly affect HBsAg-detection. By altering HBV-antigenicity and/or viral-particle maturation, they may affect full-reliability of universal diagnostic-assays for HBsAg-detection.
authors.affiliation: Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata Rome, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Svicher
authors.forename: Valeria
authors.initials: V
authors.lastname: Cento
authors.forename: Martina
authors.initials: M
authors.lastname: Bernassola
authors.forename: Maria
authors.initials: M
authors.lastname: Neumann-Fraune
authors.forename: Formijn
authors.initials: F
authors.lastname: Van Hemert
authors.forename: Mengjie
authors.initials: M
authors.lastname: Chen
authors.forename: Romina
authors.initials: R
authors.lastname: Salpini
authors.forename: Chang
authors.initials: C
authors.lastname: Liu
authors.forename: Roberta
authors.initials: R
authors.lastname: Longo
authors.forename: Michela
authors.initials: M
authors.lastname: Visca
authors.forename: Sara
authors.initials: S
authors.lastname: Romano
authors.forename: Valeria
authors.initials: V
authors.lastname: Micheli
authors.forename: Ada
authors.initials: A
authors.lastname: Bertoli
authors.forename: Caterina
authors.initials: C
authors.lastname: Gori
authors.forename: Francesca
authors.initials: F
authors.lastname: Ceccherini-Silberstein
authors.forename: Cesare
authors.initials: C
authors.lastname: Sarrecchia
authors.forename: Massimo
authors.initials: M
authors.lastname: Andreoni
authors.forename: Mario
authors.initials: M
authors.lastname: Angelico
authors.forename: Antonella
authors.initials: A
authors.lastname: Ursitti
authors.forename: Alberto
authors.initials: A
authors.lastname: Spanò
authors.forename: Jing Maria
authors.initials: JM
authors.lastname: Zhang
authors.forename: Jens
authors.initials: J
authors.lastname: Verheyen
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Cappiello
authors.forename: Carlo Federico
authors.initials: CF
authors.lastname: Perno
mesh_terms: D005260:Female; D005819:Genetic Markers; D005838:Genotype; D006509:Hepatitis B / Q000175:diagnosis* / Q000453:epidemiology; D006514:Hepatitis B Surface Antigens / Q000737:chemistry / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D008958:Models, Molecular; D009154:Mutation; D010641:Phenotype; D015995:Prevalence; D011487:Protein Conformation
chemical_list: D005819:Genetic Markers; D006514:Hepatitis B Surface Antigens