Ultra-deep sequencing reveals high prevalence and broad structural diversity of hepatitis B surface antigen mutations in a global population

Abstract

The diversity of the hepatitis B surface antigen (HBsAg) has a significant impact on the performance of diagnostic screening tests and the clinical outcome of hepatitis B infection. Neutralizing or diagnostic antibodies against the HBsAg are directed towards its highly conserved major hydrophilic region (MHR), in particular towards its “a” determinant subdomain. Here, we explored, on a global scale, the genetic diversity of the HBsAg MHR in a large, multi-ethnic cohort of randomly selected subjects with HBV infection from four continents. A total of 1553 HBsAg positive blood samples of subjects originating from 20 different countries across Africa, America, Asia and central Europe were characterized for amino acid variation in the MHR. Using highly sensitive ultra-deep sequencing, we found 72.8% of the successfully sequenced subjects (n = 1391) demonstrated amino acid sequence variation in the HBsAg MHR. This indicates that the global variation frequency in the HBsAg MHR is threefold higher than previously reported. The majority of the amino acid mutations were found in the HBV genotypes B (28.9%) and C (25.4%). Collectively, we identified 345 distinct amino acid mutations in the MHR. Among these, we report 62 previously unknown mutations, which extends the worldwide pool of currently known HBsAg MHR mutations by 22%. Importantly, topological analysis identified the “a” determinant upstream flanking region as the structurally most diverse subdomain of the HBsAg MHR. The highest prevalence of “a” determinant region mutations was observed in subjects from Asia, followed by the African, American and European cohorts, respectively. Finally, we found that more than half (59.3%) of all HBV subjects investigated carried multiple MHR mutations. Together, this worldwide ultra-deep sequencing based genotyping study reveals that the global prevalence and structural complexity of variation in the hepatitis B surface antigen have, to date, been significantly underappreciated.

Detection of in vivo hepatitis B virus surface antigen mutations—A comparison of four routine screening assays

“…These 1211 mutations in the immunodominant “a” determinant region of 844 samples were subgrouped under “first loop” (83.8%, 1015/1211) and “second loop” (16.2%, 196/1211) mutations. A summary of the specific mutations identified was also reported previously . A total of 98 (7.1%) sequenced samples revealed drug resistance‐relevant polymerase region mutations.…”

“…Details of mutations identified in the MHR “a” determinant region and their distribution by geographical region and HBV genotype have been reported previously for the 1553 HBV‐positive patient samples . Briefly, 1391 of the 1553 samples (89.5%) were successfully genotyped and analyzed for mutations in the “a” determinant region prior to HBsAg measurement (the genotype or “a” determinant region mutations could not be identified for technical reasons in the remaining 162 samples).…”

“…HBV mutations of all patient samples were detected as recently published . Briefly, PCR primers were designed to cover the S gene (defined as the region encoding amino acids 83‐227) and HBV DNA was extracted from 200 μL serum or plasma samples using the MagNA Pure 96 instrument and the MagNA Pure 96 DNA kit (Roche Applied Science).…”

Molecular epidemiology of hepatitis B virus mutants associated with vaccine escape, drug resistance and diagnosis failure

“…HBV strains carrying VEMs represent an epidemiological concern since they have the potential to infect even immunized population. Frequency of VEMs in literature covers a wide range from <5% to more than 40% . Different variables such as cohort size, prevalence of infection, time of introduction or mandatory implementation of vaccination, region analysed and viral genotypes can affect the rate of VEMs, which hinders comparison of studies.…”

“…Most epidemiological data of HBV surface and polymerase mutants have been based on studies performed in Asia or in Europe, in patients infected with genotypes B and C or A and D, respectively, with a paucity of information regarding infections with other genotypes . Particularly, information about genotype F, characteristic of Native American populations of Alaska, South and Central America and likely originated in Amerindian populations, was scarcely addressed …”