Disproportionate Distribution of HBV Genotypes A and D and the Recombinant Genotype D/E in the High and Low HBV Endemic Regions of Uganda: A Wake-Up Call for Regional Specific HBV Management

Background. Hepatitis B virus (HBV) is the leading cause of liver-related diseases. In Uganda, there is a regional disparity in the HBV burden. Our study was aimed at establishing the circulating genotypes in a low and a high endemic region to give plausible explanations for the differences in regional burden and guide the future management of the disease. Methods. A total of 200 HBsAg-seropositive subjects were recruited into the study by convenience sampling. The HBsAg Rapid Test Strip (Healgen Scientific Limited Liability Company, Houston, TX77047- USA) was used to screen for HBsAg while the Roche machine (Roche, Basel Switzerland/Abbot Technologies (USA)) was used to determine the viral load. The Chemistry Analyzer B120 (Mindray, China) was used for chemistry analysis. For HBV genotyping, total DNA was extracted from whole blood using the QIAamp® DNA extraction kit. Nested PCR amplification was performed using Platinum Taq DNA Polymerase (Invitrogen Corporation, USA) to amplify the 400 bp HBV polymerase gene. Purification of nested PCR products was performed using Purelink PCR product purification kit (Life Technologies, USA). Automated DNA sequencing was performed using BigDye Terminator v3.1 Cycle Sequencing Kit on 3130 Genetic Analyzer (Applied Biosystems, USA). The NCBI HBV genotyping tool (https://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi) was used for determination of genotype for each HBV sequence. Pearson’s chi-square, multinomial logistic regression, and Mann–Whitney U tests were used for the analysis. All the analyses were done using SPSS version 26.0 and MedCalc software version 19.1.3 at 95% CI. A p < 0.05 was considered statistically significant. Results. Majority of our study subjects were female (64.5%), youth (51.0%), and married (62.0%). Overall, genotype A was the most prevalent (46%). Genotype D and the recombinant genotype D/E were proportionately more distributed in the high endemic (38.2%) and low endemic (36.5%) regions, respectively. Genotype D was significantly more prevalent in the high endemic region and among the elderly ( p < 0.05 ). Genotype D was significantly associated with elevated viral load and direct bilirubin ( p < 0.05 ). The recombinant genotype D/E was significantly associated with elevated viral load ( p < 0.05 ). Similarly, genotype A was significantly associated with elevated AST and GGT, lowered viral load, and normal direct bilirubin levels ( p < 0.05 ). Conclusion. There is disproportionate distribution of genotypes A and D and the recombinant genotype D/E in the low and high endemic regions of Uganda. This probably could explain the differences in endemicity of HBV in our country signifying the need for regional specific HBV management and control strategies.

Mapping of dwarfing QTL of Ari1327, a semi-dwarf mutant of upland cotton

Background Upland Cotton (Gossypium hirsutum L.) has few cotton varieties suitable for mechanical harvesting. The plant height of the cultivar is one of the key features that need to modify. Hence, this study was planned to locate the QTL for plant height in a 60Co γ treated upland cotton semi-dwarf mutant Ari1327. Results Interestingly, bulk segregant analysis (BSA) and genotyping by sequencing (GBS) methods exhibited that candidate QTL was co-located in the region of 5.80–9.66 Mb at D01 chromosome in two F2 populations. Using three InDel markers to genotype a population of 1241 individuals confirmed that the offspring’s phenotype is consistent with the genotype. Comparative analysis of RNA-seq between the mutant and wild variety exhibited that Gh_D01G0592 was identified as the source of dwarfness from 200 genes. In addition, it was also revealed that the appropriate use of partial separation markers in QTL mapping can escalate linkage information. Conclusions Overwhelmingly, the results will provide the basis to reveal the function of candidate genes and the utilization of excellent dwarf genetic resources in the future.

Mapping the Genetic Regions Responsible for Key Phenology-Related Traits in the European Hazelnut

An increasing interest in the cultivation of (European) hazelnut (Corylus avellana) is driving a demand to breed cultivars adapted to non-conventional environments, particularly in the context of incipient climate change. Given that plant phenology is so strongly determined by genotype, a rational approach to support these breeding efforts will be to identify quantitative trait loci (QTLs) and the genes underlying the basis for adaptation. The present study was designed to map QTLs for phenology-related traits, such as the timing of both male and female flowering, dichogamy, and the period required for nuts to reach maturity. The analysis took advantage of an existing linkage map developed from a population of F1 progeny bred from the cross “Tonda Gentile delle Langhe” × “Merveille de Bollwiller,” consisting in 11 LG. A total of 42 QTL-harboring regions were identified. Overall, 71 QTLs were detected, 49 on the TGdL map and 22 on the MB map; among these, 21 were classified as major; 13 were detected in at least two of the seasons (stable-major QTL). In detail, 20 QTLs were identified as contributing to the time of male flowering, 15 to time of female flowering, 25 to dichogamy, and 11 to time of nut maturity. LG02 was found to harbor 16 QTLs, while 15 QTLs mapped to LG10 and 14 to LG03. Many of the QTLs were clustered with one another. The major cluster was located on TGdL_02 and consisted of mainly major QTLs governing all the analyzed traits. A search of the key genomic regions revealed 22 candidate genes underlying the set of traits being investigated. Many of them have been described in the literature as involved in processes related to flowering, control of dormancy, budburst, the switch from vegetative to reproductive growth, or the morphogenesis of flowers and seeds.

Sensitivity of the Wound Edge Gene Signature “WD14” in Responding to Clinical Change: A Longitudinal Cohort Study

Introduction: WounD14 (WD14) gene signature is a recently developed tool derived from genetic interrogation of wound edge biopsies of chronic venous leg ulcers to identify heard-to-heal wounds and enable clinicians to target aggressive therapies to promote wound healing. This study aimed to evaluate if changes in wound clinical healing status were detected by the WD14 gene signature over time as this is currently poorly understood. Material and methods: WD14 was developed through gene screening and subsequent validation in 3 patient cohorts involving 85 consecutive patients with chronic venous leg ulcers referred to a tertiary wound healing unit. Patients underwent a wound edge biopsy to interrogate for a “healing” or “non-healing” genotype. A smaller cohort (18%) underwent a second biopsy, which comprised this pilot cohort reported herein. Twelve weeks following biopsy, wounds were clinically assessed for healing status based on reduction in size and compared to WD14 genotype. Results: Sequential biopsies and WD14 scores were derived from 16 patients. WD14 signature predicted wound healing status among this cohort at either visit (32 wound edge biopsies) with a positive predictive value (PPV) of 85.2% (95% CI 74.1%-92.0%) and negative predictive value (NPV) of 80.0% (95% CI 34.2%-96.9%). A total of 6 wounds underwent altered clinical status between the 2 visits. In this cohort, WD14 has a PPV of 66.7% (95% CI 47.3%-81.7%) and NPV of 100%. Conclusion: Although the WD14 gene signature did change with wound healing status, larger studies are required to precisely clarify its role and ability to prognosticate wounds of differing clinical status over time.

miR-938 rs2505901 T>C polymorphism increases Hirschsprung disease risk: a case–control study of Chinese children

Aim: To explore the association between miR-938 rs2505901 T>C polymorphism and Hirschsprung disease (HSCR) risk in Chinese children. Materials & Methods: We conducted a case–control study in a Chinese population with 1381 cases and 1457 controls. The associated correlation strengths were assessed by adjusted odds ratios (AORs) and 95% CIs. Results: The results revealed that the rs2505901 TC and rs2505901 TC/CC genotype were related to increased HSCR risk compared to the risk contributed by the rs2505901 TT genotype. A stratification analysis showed that the rs2505901 TC/CC genotype promoted the progression of HSCR more significantly in patients with the short-segment HSCR subtype. Conclusion: Our study indicated that miR-938 rs2505901 T>C polymorphism is significantly associated with HSCR risk in Chinese children. This result needs to be confirmed with well-designed studies.

Co-detection of the measles vaccine and wild-type virus by real-time PCR: public health laboratory protocol

In rare cases vaccination with the measles virus vaccine genotype A (MeVA) may cause a vaccine reaction with clinical signs similar to infection with wild-type measles virus (MeVwt). Rapid differentiation between MeVA and MeVwt infection is important for taking adequate public health measures. Recently, a few MeVA real-time reverse-transcription quantitative PCR methods (RT-qPCRs) were described that can distinguish between MeVA and MeVwt. However, detection of MeVA does in theory not exclude infection with MeVwt. In the present study, we established a protocol for determination of co-infections with MeVA and MeVwt. To this end, MeVA RT-qPCRs were used in combination with the routine measles virus (MeV) RT-qPCR, and the results suggested that the differences between the RT-qPCR Ct values (delta Ct, ∆Ct) could be used as criteria. Subsequently, we tested samples from vaccine-associated measles cases that were confirmed by genotyping. In addition, experimental mixtures of MeVA and MeVwt were tested in different concentrations. All tested MeVA clinical samples had ∆Ct ≤3.6. The results of experimental mixtures showed a mean ∆Ct ≤2.8 for genotype A alone and >3.2 when combined with either genotype B3 or D8. The results of a receiver operator characteristic analysis indicated that the optimum ∆Ct for use as a cut-off value was 3.5, while with ∆Ct values of 2.9 and 3.7 sensitivity and specificity were respectively 1.00. Thus, ∆Ct could be used to exclude the presence of MeVwt if MeVA is detected and ∆Ct is <2.9, while ∆Ct >3.7 were highly suggestive of co-infection and ≥2.9 ∆Ct <3.7 warranted additional confirmation, such as next-generation sequencing. This RT-qPCR-based protocol could be used for the exclusion of infection with MeVwt in cases with vaccine-associated measles reaction, crucial for the timely implementation of public health prevention and control measures.

Complex structural variations in non-human primate hepatitis B virus

Background Recent genome sequence technology has revealed a novel type of genetic rearrangement referred to as complex structural variations (SVs). Previous studies have elucidated the complex SVs in human hepatitis B viruses (HBVs). In this study, we investigated the existence of complex SVs in HBVs from non-human primates (NHPs). Methods Searches for nucleotide sequences of NHP HBV were conducted using the PubMed, and genetic sequences were retrieved from databases. The candidate genetic sequences harboring complex SVs were analyzed using the CLUSTALW program and MAFFT. Additional bioinformatical analyses were performed to determine strains with complex SVs and to elucidate characteristics of NHP HBV strains. Results One hundred and fifty-four HBV strains from NHPs were identified from databases. SVs and complex SVs were observed in 11 (7.1%) strains. Three gibbon HBV (GiHBV) strains showed complex SVs consisting of an insertion and a deletion in the pre-S1 region. One GiHBV strain possessed a 6-nt insertion, which are normally specific to human HBV genotype A (HBV/A) in the Core region, and further analyses clarified that the 6-nt insertion was not caused by recombination, but rather by simple insertion. Another chimpanzee HBV strain showed complex SVs in the pre-S1 region, which were composed of human HBV/E, G-specific polymorphic SV, and an additional 6-nt insertion. Conclusions In this study, complex SVs were observed in HBV strains from NHPs, in addition to human HBV strains, as shown in previous studies. These data suggest that complex SVs could also be found in other members of hepadnaviruses, and may play a role in their genetic diversity.

Hepatitis B Transmission: Is Vertical Transmission the Major Route in Intermediate Endemic Areas? A Proof-of-Concept Study Based on Mother–Child Genotypes

The route of hepatitis B transmission is believed to be horizontal in India, though pediatric studies showed mother as source in the majority of chronic HBV (CHB) cases. We aimed at establishing the fact that mother–child transmission is the main route of acquisition by documenting genotypically identical viruses in mother–child pairs. Blood samples of consecutive children (≤18 years) with CHB and high DNA (>10,000 IU/mL) and their positive mother were collected from January 2013 to December 2015. Polymerase chain reaction (PCR) products of HBV-DNA were amplified and sequenced by using BigDye Terminator Cycle Sequencing Kit v3.1 and aligned with previously described sequences in the region of interest for genotypes A to G by using BioEdit software. Phylogenetic tree was generated using p-distance algorithm in MEGA software version 6. Genotyping of 59 (33 children and 26 mothers) subjects include genotype A in 24 (40.7%) and genotype D in 35 (59.3%). Both mother–child pair genotyping was possible in 25. The median age of 25 children (20 males) was 9 (interquartile range, IQR: 4–11). The distribution of genotypes among mother–child pairs was similar. The concordance between children and their mothers was 24 of 25 (96%). Evolutionary analyses showed significant similarities between mother and child sequences for both genotype A and D, suggesting thereby the same virus. In conclusion, mother–baby transmission seems to be the major route of acquisition of HBV in children in India and near-complete homology in genetic sequences between mother–child pairs is definite proof for that. However, a larger epidemiological study is required to substantiate our findings.