Analysis of surface antigen amino acid variations in occult hepatitis B among Indonesian blood donors

Hepatitis B virus (HBV) infection presents an example of complex host-pathogen interactions in disease progression. One of hepatitis B outcomes is occult hepatitis B infection (OBI), with its characteristically low HBV DNA titer and serologically undetectable hepatitis B surface antigen (HBsAg) - a diagnostic marker of hepatitis B. OBI poses problems in modern medicine, particularly in blood transfusion and tissue/organ transplantation. With similar transmissibility and pathogenicity as wild type HBV, occult HBV presents potential threat to the community, particularly in hepatitis B endemic areas. Indonesia as a region with moderate-to-high hepatitis B endemicity, also has a multitude of host genetic diversity. This presents an interesting field for investigation of host-pathogen interaction, which may influence the evolution of HBV in specific host background. Previous study found that from a total of 7,913 samples of regular blood donors from various regions of Indonesia, OBI was observed at approximately 10.18% prevalence rate. Research on OBI epidemiology and its molecular background will broaden our understanding of the reciprocal effects between host immunity response and pathogen evolution. The seronegativity of HBsAg in OBI may be caused by several factors, including variations of the HBsAg antigenic determinant. Screening of potentially significant HBsAg variations was performed in this study, utilizing Jameson-Wolf antigenic index calculation and protein tertiary conformation prediction to pick out amino acid variations that showed altered HBsAg antigenicity. Out of 27 novel HBsAg variations, in silico analysis highlighted 12 substitution patterns with notable antigenicity changes - most of which related to alterations of structurally important amino acids. Molecular cloning analysis confirmed the presence of 4 such mutations (sF134S, sC147Y, sS154L, and sW163R), with the existence of quasispecies population in majority of the isolates, indicating long-term chronic infection and/or co-infection of more than one HBV isolate. Other than the direct effect on HBsAg antigenicity, nucleotide changes also affect the translation of HBV polymerase gene, potentially changing the replication ability of the variant HBV. Immunoassay studies to determine the sensitivity of commercial diagnostic assays in detecting variant HBsAg were further investigated in this study. Short synthetic peptides representing the main epitopes of HBsAg - the 'a' determinant region -were tested against three HBsAg diagnostic kits commonly used in Indonesia. However, results of this immunoassay analysis should be further confirmed with development of more suitable assay method. Overall, this study underlined the significance of novel HBsAg variants in OBI and their molecular characteristics. It emphasized the importance of understanding OBI - a potential public health hazard - particularly in hepatitis B endemic regions such as Indonesia. Applications of the results of this study may lead to the development of methods to select for biologically important genomic data, employable in the improvement of hepatitis B management, including diagnostic assay formulation, therapy management, and active inhibition of viral transmission. Following this study, further analysis should be performed to confirm the biological significance of these novel HBsAg variations in both pathogen and host point of views, such as potential effect of HBV variations in altering viral replication capacity and modulating activation of host immune response

Analysis of surface antigen amino acid variations in occult hepatitis B among Indonesian blood donors

Hepatitis B virus (HBV) infection presents an example of complex host-pathogen interactions in disease progression. One of hepatitis B outcomes is occult hepatitis B infection (OBI), with its characteristically low HBV DNA titer and serologically undetectable hepatitis B surface antigen (HBsAg) - a diagnostic marker of hepatitis B. OBI poses problems in modern medicine, particularly in blood transfusion and tissue/organ transplantation. With similar transmissibility and pathogenicity as wild type HBV, occult HBV presents potential threat to the community, particularly in hepatitis B endemic areas. Indonesia as a region with moderate-to-high hepatitis B endemicity, also has a multitude of host genetic diversity. This presents an interesting field for investigation of host-pathogen interaction, which may influence the evolution of HBV in specific host background. Previous study found that from a total of 7,913 samples of regular blood donors from various regions of Indonesia, OBI was observed at approximately 10.18% prevalence rate. Research on OBI epidemiology and its molecular background will broaden our understanding of the reciprocal effects between host immunity response and pathogen evolution. The seronegativity of HBsAg in OBI may be caused by several factors, including variations of the HBsAg antigenic determinant. Screening of potentially significant HBsAg variations was performed in this study, utilizing Jameson-Wolf antigenic index calculation and protein tertiary conformation prediction to pick out amino acid variations that showed altered HBsAg antigenicity. Out of 27 novel HBsAg variations, in silico analysis highlighted 12 substitution patterns with notable antigenicity changes - most of which related to alterations of structurally important amino acids. Molecular cloning analysis confirmed the presence of 4 such mutations (sF134S, sC147Y, sS154L, and sW163R), with the existence of quasispecies population in majority of the isolates, indicating long-term chronic infection and/or co-infection of more than one HBV isolate. Other than the direct effect on HBsAg antigenicity, nucleotide changes also affect the translation of HBV polymerase gene, potentially changing the replication ability of the variant HBV. Immunoassay studies to determine the sensitivity of commercial diagnostic assays in detecting variant HBsAg were further investigated in this study. Short synthetic peptides representing the main epitopes of HBsAg - the 'a' determinant region -were tested against three HBsAg diagnostic kits commonly used in Indonesia. However, results of this immunoassay analysis should be further confirmed with development of more suitable assay method. Overall, this study underlined the significance of novel HBsAg variants in OBI and their molecular characteristics. It emphasized the importance of understanding OBI - a potential public health hazard - particularly in hepatitis B endemic regions such as Indonesia. Applications of the results of this study may lead to the development of methods to select for biologically important genomic data, employable in the improvement of hepatitis B management, including diagnostic assay formulation, therapy management, and active inhibition of viral transmission. Following this study, further analysis should be performed to confirm the biological significance of these novel HBsAg variations in both pathogen and host point of views, such as potential effect of HBV variations in altering viral replication capacity and modulating activation of host immune response

Reverse-Transcriptase Characteristics of Hepatitis B Virus Polymerase Gene in Treatment-Naïve Asymptomatic Chronic Hepatitis B Individuals

Nucleos(t)ide analogues (NUCs) remain the main treatment for chronic hepatitis B (CHB). Long-term use of NUCs significantly reduces disease progression; however, it might lead to resistance-associated mutations. We studied characteristics of polymerase gene related to NUCs resistance in naïve hepatitis B surface antigen (HBsAg)-positive individuals. The research was done at Laboratory of Hepatitis, Eijkman Institute, Jakarta Thirty eight samples were obtained and submitted for HBV DNA detection. Identification of mutations was performed by PCR-sequencing, and analyzed to obtain NUCs resistance motifs. Genotype and subtype were determined based on HBsAg sequence. Mutation of rtQ238H/N was found in 37 (97.4%) samples. Of those, 23 (62.2%) showed rtQ238H mutation, 10 (27.0%) had rtQ238N mutation, and four (10.8%) with double mutations of rtA194T and rtQ238H. Genotype B was found in 26 (68.4%), C in 11 (28.9%), and D in one (2.6%) samples. Statistically, the mutation variant of rtQ238H was associated with genotype B (p<0.001), while rtQ238N with C (p<0.001). The ayw subtype was found in 25 (65.8%), adr in 11 (28.9%), and adw in two (5.3%) samples. No mutation associated with NUCs resistance was found in most samples. This emphasizes that NUCs are still a prospective treatment in naïve CHB patients.  Mutation of rtQ238H was a variant found to be significantly associated with HBV genotype B and rtQ238N with genotype C

Improving Linkage to Care of Hepatitis C: Clinical Validation of GeneXpert R HCV Viral Load Point-of-Care Assay in Indonesia.

Hepatitis C virus (HCV) infection large-scale diagnosis and treatment are hampered by lack of a simple, rapid, and reliable point-of-care (POC) test, which poses a challenge for the elimination of hepatitis C as a public health problem. This study aimed to evaluate Cepheid Xpert R HCV Viral Load performance in comparison with the Roche Cobas R TaqMan R HCV Test using serum samples of HCV-infected patients in Indonesia. Viral load quantification was performed on 243 anti-HCV positive patients' samples using both Xpert HCV VL and Roche HCV tests, followed by HCV genotyping by reverse hybridization. Strength of the relationship between the assays was measured by Pearson correlation coefficient, while level of agreement was analyzed by Deming regression and Bland-Altman plot analysis using log10-transformed viral load values. Quantifiable viral load was detected in 180/243 (74.1%), with Xpert HCV VL sensitivity of 100% (95% CI 0.98, 1.00) and specificity of 98.4% (95% CI 0.91, 0.99) based on Roche HCV tests, while HCV genotypes were determined in 172/180 (95.6%) samples. There was a good correlation between both assays (r = 0.97, P < 0.001), overall and per genotype, with good concordance by Deming regression and a mean difference of -0.25 log10 IU/mL (95% CI -0.33, -0.18) by Bland-Altman plot analysis. Xpert HCV VL test was demonstrated as a POC platform with good performance for HCV diagnosis and treatment decision that would be beneficial for decentralized service in resource-limited areas. HCV testing sites, alongside additional GeneXpert modular systems distributed toward the fight against COVID-19, could ensure some continuity, once this pandemic is controlled

HBV sequences used in this study.

<p>^† Details of the GenBank Accession Numbers for all HBV sequences are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132533#pone.0132533.s004" target="_blank">S1 Table</a>.</p><p>^‡ Five HBV/C sequences initially used in phylogenetic tree construction representing HBV/C7, C9, C10, C15, and C16 were not used in nucleotide divergence analysis because only single complete genome isolates were available for each of these subgenotypes.</p><p>^§ Core immune epitope analysis used sequences that cover the C gene region. Compared to the sequences used in the surface immune epitope analysis, only 16 HBV/C Asia sequences were used again in the core immune epitope analysis, while all S gene sequences from HBV/C Papua Pacific and the 87 HBV/C Indonesia of this study did not qualify for the core immune epitope analysis.</p><p>HBV sequences used in this study.</p

Distribution of HBsAg subtypes and HBV genotypes/subgenotypes of 271 HBV/C isolates according to their country/geographical origins in East/Southeast Asia and Papua-Pacific.

<p>^{# including 37 published complete genome sequences and 87 newly generated in this study; N. Caledonia: New Caledonia; PNG: Papua New Guinea.}</p><p>Distribution of HBsAg subtypes and HBV genotypes/subgenotypes of 271 HBV/C isolates according to their country/geographical origins in East/Southeast Asia and Papua-Pacific.</p

HBcAg amino acid motifs in B and T-cell epitopes of Asia and Papua-Pacific HBV/C isolates.

<p>For the sake of clarity, this figure was not drawn to scale. Among 15 amino acid positions examined within HBcAg immune epitopes of 143 isolates, we identified I/V at position c59 as the essential variation that classified HBV/C subgenotypes into two major clusters, the Asian and the Papua-Pacific (p-value <0.001; data not shown). HBV/C4 and C14 showed similar variation in most amino acids examined, with C4 and C14 having cI59 and cV59, respectively.</p

Phylogenetic tree of HBV/C isolates from different countries in East and Southeast Asia, and Papua-Pacific.

<p>A Bayesian phylogenetic tree analysis based on complete genome sequences showed that isolates from various subgenotypes (C1-C16) are clearly grouped into two major clusters, consistent with their geographical origins. Seven HBV/C subgenotypes (C1, C2, C5, C7, C8, C9, and C10) from East and Southeast Asia, and one (C14) from Papua (<i>light highlight</i>) were well-separated from those six subgenotypes (C6, C11, C12, C13, C15, and C16) from Papua, and from one subgenotype (C3) from Pacific, the more east region of the Papua (<i>dark highlight</i>). Although the root of subgenotype C3 phylogenetically is distanced from the subgenotypes of Papua, the isolate geographic origin, the immune epitope characteristics of surface and core proteins, and the HBsAg subtype gradient distribution showed these HBV/C3 isolates to be close to Papua subgenotypes. Therefore, the Papua and the Pacific subgenotypes are classified together into Papua-Pacific type. The diversification of the Asian type from the Papua-Pacific type started from Papua of Indonesia to the east. The other subgenotype, HBV/C4, was distanced from other subgenotypes. In this analysis, one strain (GQ358157) from Papua reported as C6 in our previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132533#pone.0132533.ref023" target="_blank">23</a>] grouped into C12. We redefine this strain as a member of HBV/C12.</p

Distribution of HBV/C subtypes in the East and Southeast Asia and the Papua-Pacific.

<p>This study identified a west-to-east gradient in the distribution of HBsAg subtypes with <i>adrq+</i> (<i>red</i>) prominent in East-Southeast Asia and <i>adrq-</i> (<i>pink</i>) in the Pacific region (Vanuatu, Fiji, Tonga, and Kiribati). Interestingly, together with <i>adrq+</i>, <i>adrq-</i>indeterminate sA159/sA177 and a new pattern of <i>adrq-</i>indeterminate sV159/sV177 identified in this study were found in Papua and PNG, respectively, suggesting that the molecular admixture of HBV/C, particularly for subtype evolution, occurred in Papua and PNG with both <i>adrq-</i>indeterminate forms (<i>yellow</i>) as the transitional patterns.</p