Endemic HBV among hospital in-patients in Bangladesh, including evidence of occult infection

Bangladesh is one of the top-ten most heavily burdened countries for viral hepatitis, with hepatitis B (HBV) infections responsible for the majority of cases. Recombinant and occult HBV infections (OBI) have been reported previously in the region. We investigated an adult fever cohort (n=201) recruited in Dhaka, to determine the prevalence of HBV and OBI. A target-enrichment deep sequencing pipeline was applied to samples with HBV DNA >3.0 log10 IU ml−1. HBV infection was present in 16/201 (8 %), among whom 3/16 (19 %) were defined as OBI (HBsAg-negative but detectable HBV DNA). Whole genome deep sequences (WGS) were obtained for four cases, identifying genotypes A, C and D. One OBI case had sufficient DNA for sequencing, revealing multiple polymorphisms in the surface gene that may contribute to the occult phenotype. We identified mutations associated with nucleos(t)ide analogue resistance in 3/4 samples sequenced, although the clinical significance in this cohort is unknown. The high prevalence of HBV in this setting illustrates the importance of opportunistic clinical screening and DNA testing of transfusion products to minimise OBI transmission. WGS can inform understanding of diverse disease phenotypes, supporting progress towards international targets for HBV elimination.

Low Genetic Diversity of Hepatitis B Virus Surface Gene amongst Australian Blood Donors

Variants in the small surface gene of hepatitis B virus (HBV), which codes for viral surface antigen (HBsAg), can affect the efficacy of HBsAg screening assays and can be associated with occult HBV infection (OBI). This study aimed to characterise the molecular diversity of the HBV small surface gene from HBV-reactive Australian blood donors. HBV isolates from 16 HBsAg-positive Australian blood donors’ plasma were sequenced and genotyped by phylogenies of viral coding genes and/or whole genomes. An analysis of the genetic diversity of eight HBV small surface genes from our 16 samples was conducted and compared with HBV sequences from NCBI of 164 international (non-Australian) blood donors. Genotypes A–D were identified in our samples. The region of HBV small surface gene that contained the sequence encoding the ‘a’ determinant had a greater genetic diversity than the remaining part of the gene. No escape mutants or OBI-related variants were observed in our samples. Variant call analysis revealed two samples with a nucleotide deletion leading to truncation of polymerase and/or large/middle surface amino acid sequences. Overall, we found that HBV small surface gene sequences from Australian donors demonstrated a lower level of genetic diversity than those from non-Australian donor population included in the study.

Endemic hepatitis B virus (HBV) among hospital in-patients in Bangladesh, including evidence of occult infection

ABSTRACTBangladesh is one of the world’s top ten burdened countries for viral hepatitis. We investigated an adult fever cohort (n=201) recruited in Dhaka, to determine the prevalence of hepatitis B virus (HBV) infection and to identify cases of occult hepatitis B infection (OBI). HBV exposure (anti-HBc) was documented in 72/201 (36%), and active HBV infection in 16/201 (8%), among whom 3 were defined as OBI (defined as detectable HBV DNA but negative HBsAg). Applying a target-enrichment sequencing pipeline to samples with HBV DNA >3.0log10 IU/ml, we obtained deep whole genome sequences for four cases, identifying genotypes A, C and D. Polymorphisms in the surface gene of the OBI case may account for the negative HBsAg status. We identified mutations associated with nucleos(t)ide analogue resistance, although the clinical significance in this cohort is not known. The high prevalence of HBV in this setting highlights the benefits of offering screening in hospital patients and the importance of HBV DNA testing of transfusion products to reduce the risk of transmission. In order to work towards international Sustainable Development Goal targets for HBV elimination, increased investment is required for diagnosis, treatment and prevention in Bangladesh.

A25 Impact of polymorphism in the hepatitis B surface gene on human leukocyte antigen (HLA) class II

There is still no cure for chronic hepatitis B virus infection (CHBV), a major cause of liver cancers and related malignancies. Elucidating the role of CD4+ T-helper cells in activating immunological responses that clear antigenic peptides during primary HBV infection holds a potential strategy for developing potent vaccines. Since the strength of CD4+ T cell responses is dictated by binding of viral epitopes to class-II human leukocyte antigens (HLAs), we hypothesize that the quality of immunological responses in CHBV patients is influenced by host genetics and HBV genotypes. Here, ninety-two non-recombinant complete HBV surface-gene proteins (PreS1/S) from Botswana were sequenced (genotype A 44(47.8%); D 48(52.2%)) and 15-mer binding epitopes restricted to nine HLA-class II molecules (DRB5/1) were mapped in silico. The HLAs used have high population coverage in Botswana. The total predicted epitopes per HLA were 94-(genotype A) and 105-(genotype D) for PreS1, 42 (A and D) for PreS2, and 105 (A and D) for S. Epitope densities (binding peptides to total epitopes) were 3 per cent and 6 per cent (PreS1A&D), 4 per cent and 2 per cent (PreS2A&D), and 23 per cent and 22 per cent (S1A&D). SA&D proteins had most polytopes: CPGYRWMCLRRFII66-81, PGYRWMCLRRFIIF67-82, GYRWMCLRRFIIFL68-83, and YRWMCLRRFIIFLF69-84 binding to 5 (55.6%) HLAs (DRB1*0101/0701/1101/1501 and DRB5*0101) used. HLA-DRB*0101 bound the most epitopes, and the least were bound by HLA-DRB*0302/0701/0401 for both genotypes. PreS1D polytope: PAFRANTANPDWDFN32-46 binds to DRB1*0101/0401/1302 and PreS2 polytopes: TAFHQALQDPRVRG6-19 and AFHQALQDPRVRGL7-20 bind to DRB1*010/1501 alleles. Non-synonymous mutations impair peptide-HLA binding when assessed as combinations of > 2. The least active HLAs may be associated with CHBV and vice-versa for HBV clearance, thus the algorithm may be used to predict HBV prognosis for different haplotypes. The results favor the use of epitopes from S protein as broad genotype vaccine. This study highlights the need to explore further the mechanisms of PreS1 and its effect on the immune system.

Multiple layers of cryptic genetic variation underlie a yeast complex trait

Cryptic genetic variation may be an important contributor to heritable traits, but its extent and regulation are not fully understood. Here, we investigate the cryptic genetic variation underlying a Saccharomyces cerevisiae colony phenotype that is typically suppressed in a cross of the lab strain BY4716 (BY) and a derivative of the clinical isolate 322134S (3S). To do this, we comprehensively map the trait's genetic basis in the BYx3S cross in the presence of three different genetic perturbations that enable its expression. This allows us to detect and compare the specific loci harboring cryptic genetic variants that interact with each perturbation. In total, we identify 21 loci, all but one of which interacts with just a subset of the perturbations. Beyond impacting which loci contribute to the trait, the genetic perturbations also influence the extent of additivity, epistasis, and genotype-environment interaction among the detected loci. Additionally, we show that the single locus interacting with all three perturbations corresponds to the coding region of the cell surface gene FLO11. Nearly all of the other loci influence FLO11 transcription in cis or trans. However, the perturbations reveal cryptic genetic variation in different pathways and sub-pathways upstream of FLO11, suggesting that multiple layers of cryptic genetic variation with highly contextual effects underlie the trait. Our work demonstrates an abundance of cryptic genetic variation in transcriptional regulation and illustrates how this cryptic genetic variation complicates efforts to study the relationship between genotype and phenotype.