Molecular and Serological Characterization of Hepatitis B Virus (HBV)-Positive Samples with Very Low or Undetectable Levels of HBV Surface Antigen

Background: Gaps remain in the detection of nucleic acid test (NAT) yield and occult hepatitis B virus (HBV) infection (OBI) by current HBV surface antigen (HBsAg) assays. The lack of detection may be due to HBsAg levels below current assay detection limits, mutations affecting HBsAg assays or HBsAg levels, or the masking of HBsAg by antibody to HBsAg (anti-HBs). In this study, we evaluate the incremental detection of NAT yield and OBI from five diverse geographic areas by an improved sensitivity HBsAg assay and characterize the samples relative to the viral load, anti-HBs status, and PreS1–S2–S mutations. Included is a comparison population with HBV DNA levels comparable to OBI, but with readily detectable HBsAg (High Surface–Low DNA, HSLD). Methods: A total of 347 samples collected from the USA, South Africa, Spain, Cameroon, Vietnam, and Cote D’Ivoire representing NAT yield (HBsAg(−), antibody to HBV core antigen (anti-HBc)(−), HBV DNA(+), N = 131), OBI (HBsAg(−), anti-HBc(+), HBV DNA(+), N = 188), and HSLD (HBsAg(+), anti-HBc(+), HBV DNA(+), N = 28) were tested with ARCHITECT HBsAg NEXT (HBsAgNx) (sensitivity 0.005 IU/mL). The sequencing of the PreS1–S2–S genes from a subset of 177 samples was performed to determine the genotype and assess amino acid variability, particularly in anti-HBs(+) samples. Results: HBsAgNx detected 44/131 (33.6%) NAT yield and 42/188 (22.3%) OBI samples. Mean HBV DNA levels for NAT yield and OBI samples were lower in HBsAgNx(−) (50.3 and 25.9 IU/mL) than in HBsAgNx(+) samples (384.1 and 139.5 IU/mL). Anti-HBs ≥ 10 mIU/mL was present in 28.6% HBsAgNx(+) and 45.2% HBsAgNx(−) OBI, and in 3.6% HSLD samples. The genotypes were A1, A2, B, C, D, E, F, and H. There was no significant difference between HBsAgNx(−) and HBsAgNx(+) in the proportion of samples harboring substitutions or in the mean number of substitutions per sample in PreS1, PreS2, or S for the NAT yield or OBI (p range: 0.1231 to >0.9999). A total of 21/27 (77.8%) of HBsAgNx(+) OBI carried S escape mutations, insertions, or stop codons. HSLD had more PreS1 and fewer S substitutions compared to both HBsAgNx(−) and HBsAgNx(+) OBI. Mutations/deletions associated with impaired HBsAg secretion were observed in the OBI group. Conclusions: HBsAgNx provides the improved detection of NAT yield and OBI samples. Samples that remain undetected by HBsAgNx have exceptionally low HBsAg levels below the assay detection limit, likely due to low viremia or the suppression of HBsAg expression by host and viral factors.

Identification and Characterization of DAMs Mutations Associated With Early Blooming in Sweet Cherry, and Validation of DNA-Based Markers for Selection

Dormancy release and bloom time of sweet cherry cultivars depend on the environment and the genotype. The knowledge of these traits is essential for cultivar adaptation to different growing areas, and to ensure fruit set in the current climate change scenario. In this work, the major sweet cherry bloom time QTL qP-BT1.1m (327 Kbs; Chromosome 1) was scanned for candidate genes in the Regina cv genome. Six MADS-box genes (PavDAMs), orthologs to peach and Japanese apricot DAMs, were identified as candidate genes for bloom time regulation. The complete curated genomic structure annotation of these genes is reported. To characterize PavDAMs intra-specific variation, genome sequences of cultivars with contrasting chilling requirements and bloom times (N = 13), were then mapped to the ‘Regina’ genome. A high protein sequence conservation (98.8–100%) was observed. A higher amino acid variability and several structural mutations were identified in the low-chilling and extra-early blooming cv Cristobalina. Specifically, a large deletion (694 bp) upstream of PavDAM1, and various INDELs and SNPs in contiguous PavDAM4 and -5 UTRs were identified. PavDAM1 upstream deletion in ‘Cristobalina’ revealed the absence of several cis-acting motifs, potentially involved in PavDAMs expression. Also, due to this deletion, a non-coding gene expressed in late-blooming ‘Regina’ seems truncated in ‘Cristobalina’. Additionally, PavDAM4 and -5 UTRs mutations revealed different splicing variants between ‘Regina’ and ‘Cristobalina’ PavDAM5. The results indicate that the regulation of PavDAMs expression and post-transcriptional regulation in ‘Cristobalina’ may be altered due to structural mutations in regulatory regions. Previous transcriptomic studies show differential expression of PavDAM genes during dormancy in this cultivar. The results indicate that ‘Cristobalina’ show significant amino acid differences, and structural mutations in PavDAMs, that correlate with low-chilling and early blooming, but the direct implication of these mutations remains to be determined. To complete the work, PCR markers designed for the detection of ‘Cristobalina’ structural mutations in PavDAMs, were validated in an F2 population and a set of cultivars. These PCR markers are useful for marker-assisted selection of early blooming seedlings, and probably low-chilling, from ‘Cristobalina’, which is a unique breeding source for these traits.

Amino acid variability, tradeoffs and optimality in human diet

While the quality of fat (e.g. saturated/unsaturated) and carbohydrate (e.g. whole grain/simple sugars) intake has been of great interest, less attention has been made to the type of protein and resulting amino acid intake profiles in human diets. Studies at the molecular level however demonstrate that dietary amino acid intake produces substantial effects on health and disease such as cancer by modulating metabolism. How these effects may manifest in human food consumption and dietary patterns is unknown. We developed a series of algorithms to map, characterize and model the landscape of amino acid content in human food, dietary patterns, and individual consumption including relations to health status, covering over 2,000 foods, ten dietary patterns, and over 30,000 dietary records. We found that the type of amino acids contained in foods and human consumption is highly dynamic with variability far exceeding that of fat and carbohydrate. Some amino acids positively associate with diseases such as obesity while others contained in the same food negatively link to disease. Using linear programming and machine learning, we show that these health trade-offs among can be accounted to satisfy biochemical constraints in food and human eating patterns to construct a Pareto front in dietary practice, a means of achieving optimality in the face of tradeoffs that are commonly considered in economic and evolutionary theories. Thus this study may enable the design of human protein quality intake guidelines based on a quantitative framework.

Biochemical features of parvovirus B19 genovariant 1a2 dominating during the incidence rise in Belarus

Two genovariants (1a1 and 1a2) are distinguished among Human parvovirus B19 (B19P) of subgenotype 1a, of which 1a2 was predominantly distributed during the incidence rise in Belarus. The aim of this study was a comparative analysis of the amino acid variability and of the mutational pressure directions in different parts of the genome between genovariants 1a1 and 1a2.The analysis of the consensus amino acid sequences of two genovariants and the three-dimensional structure models of protein fragments was carried out. In total, two unique amino acid substitutions in the main non-structural protein NS1 of 1a2 were found (I181M and E114G), one of which E114G is close to the DNA-binding domain (OBD) responsible for attachment to the replication origin site and can affect the rate of virus replication and transcription. Three unique amino acid substitutions were found in the structural polypeptide VP of 1a2: V30L, S98N, and N533S. Two of them are located in the most immunogenic region VP1u and can contribute to the escape from immune response. The investigation of the mutational pressure direction revealed a decrease in the frequency of G to T transversions in the second reading frame of 1a2, which reflects a higher transcription rate as a result of amino acid substitution in the OBD protein.The differences revealed between the genetic variants of subgenotype 1a B19P both in the antigenic sites and in the replication and transcription system can provide an increased “fitness” for the genetic variant 1a2 and explain its predominant distribution during the incidence rise.

SAT-412 Genetic Susceptibility to Graves’ Disease Conferred by HLA-DQβ1 Position 57

Introduction: Human leukocyte antigen (HLA) locus is the major genetic risk factor for autoimmune endocrine diseases. The amino acid variability at the HLA-DQβ1 position 57 encoded by both DQB1 alleles was identified as critical risk residue for type 1 diabetes. We therefore investigated this amino acid residue at HLA-DQβ1 position 57 in Graves’ disease (GD). Subjects and methods: DNA samples were obtained from 572 healthy controls (HC, 262 females/310 males) and 299 patients with GD (255 females/44 males) and genotyped for HLA-DQB1 using a sequence specific primer (SSP, 13 primer pairs) approach. The PCR amplified products were analyzed by gel electrophoresis. The HLA-DQB1 alleles were defined with their corresponding amino acid residues at the DQß chain positon 57 as follows: Ala57 (DQB1*0201,*0302), Asp57 (DQB1*0301,*0303,*0401,*0402,*0503,*0601,*0602*,0603) Val57 (DQB1*0501,*0604) and Ser57 (DQB1*0502). Finally, the frequencies for amino acid variabilities and their combinations (Ala/Ala, Ala/non-Ala and non-Ala/non-Ala) were calculated and compared by Pearson-Mantel-Haenszel Chi-squared test. Results: The presence of Ala57 was significantly more frequent in patients with GD compared to HC (25% vs. 39%, OR: 1.77; p=7x10-4), whereas Val57 was less frequent (19% vs. 12%, OR: 0.59; p=0.04). In contrast, no difference in the distribution between GD and HC concerning the amino acids Asp57 and Ser57 was observable. Similar results were found in female (Ala57 23% vs. 38%, OR:2.01; p=1.2x10-3 and Val57 19% vs. 13%, OR:0.35; p=1.2x10-4) but not in males (p >0.5). Furthermore, homozygous HLA-DQB1 Ala (8% vs. 15%, OR:1.98; p=5x10-3) as well as heterozygous HLA-DQB1 Ala/non-Ala (34% vs. 45%, OR:1.58; pc=5x10-3) were significantly more prevalent, while non-Ala/non-Ala (58% vs. 40%, OR:0.49 pc=3x5x10-6) was found to be less frequent in patients than in HC. By gender stratification there was a significant difference between female patients with GD and female HC in the distribution of the mentioned amino acid combinations (Ala p=4.5x10-3, Ala/non-Ala pc=0.02, non-Ala/non-Ala p=1x10-5). Conclusion: We identify a strongly predisposing role for HLA-DQβ1 Ala5 7to GD, particularly in women, highlighting the gender specific disease risk. Immunogenetic testing may pave the way towards personalized treatment.

HDV Can Constrain HBV Genetic Evolution in HBsAg: Implications for the Identification of Innovative Pharmacological Targets

Chronic HBV + HDV infection is associated with greater risk of liver fibrosis, earlier hepatic decompensation, and liver cirrhosis hepatocellular carcinoma compared to HBV mono-infection. However, to-date no direct anti-HDV drugs are available in clinical practice. Here, we identified conserved and variable regions in HBsAg and HDAg domains in HBV + HDV infection, a critical finding for the design of innovative therapeutic agents. The extent of amino-acid variability was measured by Shannon-Entropy (Sn) in HBsAg genotype-d sequences from 31 HBV + HDV infected and 62 HBV mono-infected patients (comparable for demographics and virological-parameters), and in 47 HDAg genotype-1 sequences. Positions with Sn = 0 were defined as conserved. The percentage of conserved HBsAg-positions was significantly higher in HBV + HDV infection than HBV mono-infection (p = 0.001). Results were confirmed after stratification for HBeAg-status and patients’ age. A Sn = 0 at specific positions in the C-terminus HBsAg were correlated with higher HDV-RNA, suggesting that conservation of these positions can preserve HDV-fitness. Conversely, HDAg was characterized by a lower percentage of conserved-residues than HBsAg (p < 0.001), indicating higher functional plasticity. Furthermore, specific HDAg-mutations were significantly correlated with higher HDV-RNA, suggesting a role in conferring HDV replicative-advantage. Among HDAg-domains, only the virus-assembly signal exhibited a high genetic conservation (75% of conserved-residues). In conclusion, HDV can constrain HBsAg genetic evolution to preserve its fitness. The identification of conserved regions in HDAg poses the basis for designing innovative targets against HDV-infection.

Glycoprotein 3 of Porcine Reproductive and Respiratory Syndrome Virus Exhibits an Unusual Hairpin-Like Membrane Topology

ABSTRACTGlycoprotein 3 (GP3) of the arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) consists of a cleaved signal peptide, a highly glycosylated domain, a short hydrophobic region, and an unglycosylated C-terminal domain. GP3 is supposed to form a complex with GP2 and GP4 in virus particles, but secretion of GP3 from cells has also been reported. We analyzed the membrane topology of GP3 from various PRRSV strains. A fraction of the protein is secreted from transfected cells, GP3 from PRRSV-1 strains to a greater extent than GP3 from PRRSV-2 strains. This secretion behavior is reversed after exchange of the variable C-terminal domain. A fluorescence protease protection assay shows that the C terminus of GP3, fused to green fluorescent protein (GFP), is resistant to proteolytic digestion in permeabilized cells. Furthermore, glycosylation sites inserted into the C-terminal part of GP3 are used. Both experiments indicate that the C terminus of GP3 is translocated into the lumen of the endoplasmic reticulum. Deletion of the conserved hydrophobic region greatly enhances secretion of GP3, and fusion of this domain to GFP promotes membrane anchorage. Bioinformatics suggests that the hydrophobic region forms an amphipathic helix. Accordingly, exchanging only a few amino acids in its hydrophilic face prevents secretion of GP3 and in its hydrophobic face enhances it. Exchanging the latter amino acids in the context of the viral genome did not affect release of virions, but released particles were not infectious. In sum, GP3 exhibits an unusual hairpin-like membrane topology that might explain why a fraction of the protein is secreted.IMPORTANCEPRRSV is the most important pathogen in the pork industry. It causes persistent infections that lead to reduced weight gain of piglets; highly pathogenic strains even kill 90% of an infected pig population. PRRSV cannot be eliminated from pig farms by vaccination due to the large amino acid variability between the existing strains, especially in the glycoproteins. Here, we analyzed basic structural features of GP3 from various PRRSV strains. We show that the protein exhibits an unusual hairpin-like membrane topology; membrane anchoring might occur via an amphipathic helix. This rather weak membrane anchor explains why a fraction of the protein is secreted from cells. Interestingly, PRRSV-1 strains secrete more GP3 than PRRSV-2. We speculate that secreted GP3 plays a role during PRRSV infection of pigs: it might serve as a decoy to distract antibodies away from virus particles.

Glycoprotein 3 of porcine reproductive and respiratory syndrome virus exhibits an unusual hairpin-like membrane topology

ABSTRACTThe glycoprotein GP3 of the Arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) consists of a cleaved signal peptide, a highly glycosylated domain, a short hydrophobic region and an unglycosylated C-terminal domain. GP3 is supposed to form a complex with GP2 and GP4 in virus particles, but secretion of GP3 from cells has also been reported.We analyzed the membrane topology of GP3 from various PRRSV strains. A fraction of the protein is secreted from transfected cells; GP3 from PRRSV-1 strains to a greater extent than GP3 from PRRSV-2 strains. This secretion behavior is reversed after exchange of the variable C-terminal domain. A fluorescence protease protection assay shows that the C-terminus of GP3, fused to GFP, is resistant against proteolytic digestion in permeabilized cells. Furthermore, glycosylation sites inserted into the C-terminal part of GP3 are used. Both experiments indicate that the C-terminus of GP3 is translocated into the lumen of the endoplasmic reticulum. Deletion of the conserved hydrophobic region greatly enhances secretion of GP3 and fusion of this domain to GFP promotes membrane anchorage. Bioinformatics suggests that the hydrophobic region might form an amphipathic helix. Accordingly, exchanging only a few amino acids in its hydrophilic face prevents and in its hydrophobic face enhances secretion of GP3. Exchanging the latter amino acids in the context of the viral genome did not affect release of virions, but released particles were not infectious. In sum, GP3 exhibits an unusual hairpin-like membrane topology that might explain why a fraction of the protein is secreted.IMPORTANCEThe porcine reproductive and respiratory syndrome virus (PRRSV) is the most important pathogen in the pork industry. It causes persistent infections that lead to reduced weight gain of piglets; highly pathogenic strains even kill 90% of an infected pig population. PRRSV cannot be eliminated from pig farms by vaccination due to the large amino acid variability between the existing strains, especially in the glycoproteins. Here we analyzed basic structural features of glycoprotein 3 (GP3) from various PRRSV strains. We show that the protein exhibits an unusual hairpin-like membrane topology; membrane anchoring might occur via an amphipathic helix. This rather weak membrane anchor explains why a fraction of the protein is secreted from cells. Interestingly, PRRSV-1 strains secrete more GP3 than PRRSV-2. We speculate that secreted GP3 might play a role during PRRSV infection of pigs; it might serve as a decoy to distract antibodies away from virus particles.