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.
Bibliographical noteFunding Information:
MG, RB, DN, and GW are employees of Roche Diagnostics International Ltd, Rotkreuz, Switzerland. AW and CR are employees of Roche Diagnostics GmbH, Penzberg, Germany. WEK, MN, KH, PG, and EB are employees of Bioscientia GmbH, which received research funding from Roche Diagnostics. PTTT is an employee of Medic Medical Center, Ho Chi Minh City, Vietnam, which received research funding from Roche Diagnostics. BHH is an employee of Ho Chi Minh City University Medical Center, Ho Chi Minh City, Vietnam, which received research funding from Roche Diagnostics. MS and CWS are employees of Groote Schuur Hospital, Cape Town, South Africa, which received research funding from Roche Diagnostics. GB and GBG are employees of Infectious Diseases and Viral Hepatitis Unit, Second University of Naples, Italy, which received research funding from Roche Diagnostics. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
© 2017 Gencay et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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