Phylogeography of hepatitis B: The role of Portugal in the early dissemination of HBV worldwide

In Portugal, the genetic diversity, origin of HBV, and the Portuguese role in the dissemination of HBV worldwide were never investigated. In this work, we studied the epidemic history and transmission dynamics of HBV genotypes that are endemic in Portugal. HBV pol gene was sequenced from 130 patients followed in Lisbon. HBV genotype A (HBV/A) was the most prevalent (n=54, 41.5%), followed by D [HBV/D; (n=44, 33.8%)], and E [HBV/E; (n=32, 24.6%)]. Spatio-temporal evolutionary dynamics was reconstructed in BEAST using a Bayesian Markov Chain Monte Carlo method, with a GTR nucleotide substitution model, an uncorrelated lognormal relaxed molecular clock model, a Bayesian skyline plot, and a continuous diffusion model. HBV/D4 was the first subgenotype to be introduced in Portugal around 1857 (HPD 95% 1699-1931) followed by HBV/D3 and A2 a few decades later. HBV/E and HBV/A1 were introduced in Portugal later, almost simultaneously. Our results also indicate a very important role of Portugal in the exportation of HBV/D4 and A2 to Brazil and Cape Verde, respectively, at the beginning of the XX century. This work clarifies the epidemiological history of HBV in Portugal and shows that Portugal had an important role in the global spread of this virus.

More DNA and RNA of HBV SP1 splice variants are detected in genotypes B and C at low viral replication

HBV produces unspliced and spliced RNAs during replication. Encapsidated spliced RNA is converted into DNA generating defective virions that are detected in plasma and associated with HCC development. Herein we describe a quantitative real-time PCR detection of splice variant SP1 DNA/RNA in HBV plasma. Three PCR primers/probe sets were designed detecting the SP1 variants, unspliced core, or X gene. Plasmids carrying the three regions were constructed for the nine HBV genotypes to evaluate the three sets, which were also tested on DNA/RNA extracted from 193 HBV plasma with unknown HCC status. The assay had an LOD of 80 copies/ml and was equally efficient for detecting all nine genotypes and three targets. In testing 84 specimens for both SP1 DNA (77.4%) and RNA (82.1%), higher viral loads resulted in increased SP1 levels. Most samples yielded < 1% of SP1 DNA, while the average SP1 RNA was 3.29%. At viral load of ≤ 5 log copies/ml, the detectable SP1 DNA varied by genotype, with 70% for B, 33.3% for C, 10.5% for E, 4% for D and 0% for A, suggesting higher levels of splicing in B and C during low replication. At > 5 log, all samples regardless of genotype had detectable SP1 DNA.

Variability in the response of HBV D-subgenotypes to antiviral therapy: designing pan D-subgenotypic reverse transcriptase inhibitors

Nucleos(t)ide analogues entecavir (ETV ) and tenofovir disoproxil fumarate (TDF ) are recommended as first - line monotherapies for chronic hepatitis B (CHB). Multiple HBV genotypes/subgenotypes have been described, but their impact on treatment response remains largely elusive. We investigated the effectiveness of ETV/TDF on HBV/D-subgenotypes, D1/D2/D3/D5, studied the structural/functional differences in subgenotype-specific reverse transcriptase (RT) domains of viral polymerase and identified novel molecules with robust inhibitory activity on various D-subgenotypes. Transfection of Huh7 cells with full-length D1/D2/D3/D5 and in vitro TDF/ETV susceptibility assays demonstrated that D1/D2 had greater susceptibility to TDF/ETV while D3/D5 exhibited poorer response. Additionally, HBV load was substantially reduced in TDF-treated CHB patients carrying D1/D2 but minimally reduced in D3/D5-infected patients. Comparison of RT sequences of D-subgenotypes led to identification of unique subgenotype-specific residues and molecular modeling/docking/simulation studies depicted differential bindings of TDF/ETV to the active site of their respective RTs. Replacement of signature residues in D3/D5 HBV clones with corresponding amino acids seen in D1/D2 improved their susceptibility to TDF/ETV. Using high throughput virtual screening, we identified N(9)-[3 - fluoro - 2-(phosphonomethoxy)propyl] (FPMP) derivatives of purine bases, including N 6 -substituted ( S )-FPMP derivative of 2,6-diaminopurine (DAP) (OB-123-VK), as potential binders of RT of different D-subgenotypes. We synthesized ( S )-FPMPG prodrugs (FK-381-FEE/FK-381-SEE/FK-382) and tested their effectiveness along with OB-123-VK. Both OB-123-VK and FK-381-FEE exerted similar antiviral activities against all D-subgenotypes, although FK-381-FEE was more potent. Our study highlighted the natural variation in therapeutic response of D1/D2/D3/D5 and emphasized the need for HBV subgenotype determination before treatment. Novel molecules described here could benefit future design/discovery of pan-D-subgenotypic inhibitors. Importance: Current treatment of chronic hepatitis B relies heavily on nucleotide/nucleoside analogs in particular, tenofovir disoproxil fumarate (TDF) and entecavir (ETV) to keep HBV replication under control and prevent end-stage liver diseases. However, it was unclear whether the therapeutic effects of TDF/ETV differ among patients infected with different HBV genotypes and subgenotypes. HBV genotype D is the most widespread of all HBV genotypes and multiple D-subgenotypes have been described. We here report that different subgenotypes of HBV genotype-D exhibit variable response towards TDF and ETV and this could be attributed to naturally occurring amino acid changes in the reverse transcriptase domain of the subgenotype-specific polymerase. Further, we identified novel molecules and also synthesized prodrugs that are equally effective on different D-subgenotypes and could facilitate management of HBV/D-infected patients irrespective of D-subgenotype.

HERC5 E3 ligase mediates ISGylation of hepatitis B virus X protein to promote viral replication

Ubiquitin and ubiquitin-like protein modification play important roles in modulating the functions of viral proteins in many viruses. Here we demonstrate that hepatitis B virus (HBV) X protein (HBx) is modified by ISG15, which is a type I IFN-inducible, ubiquitin-like protein; this modification is called ISGylation. Immunoblot analyses revealed that HBx proteins derived from four different HBV genotypes accepted ISGylation in cultured cells. Site-directed mutagenesis revealed that three lysine residues (K91, K95 and K140) on the HBx protein, which are well conserved among all the HBV genotypes, are involved in acceptance of ISGylation. Using expression plasmids encoding three known E3 ligases involved in the ISGylation to different substrates, we found that HERC5 functions as an E3 ligase for HBx-ISGylation. Treatment with type I and type III IFNs resulted in the limited suppression of HBV replication in Hep38.7-Tet cells. When cells were treated with IFN-α, silencing of ISG15 resulted in a marked reduction of HBV replication in Hep38.7-Tet cells, suggesting a role of ISG15 in the resistance to IFN-α. In contrast, the silencing of USP18 (an ISG15 de-conjugating enzyme) increased the HBV replication in Hep38.7-Tet cells. Taken together, these results suggest that the HERC5-mediated ISGylation of HBx protein confers pro-viral functions on HBV replication and participates in the resistance to IFN-α-mediated antiviral activity.

A CRISPR-Cas12b–Based Platform for Ultrasensitive, Rapid, and Highly Specific Detection of Hepatitis B Virus Genotypes B and C in Clinical Application

Hepatitis B virus (HBV) is one of the most dangerous and prevalent agents that causes acute and chronic liver diseases in humans. Genotyping plays an important role in determining clinical outcomes and response to antiviral treatment in HBV–infected patients. Here, we first devised a CRISPR–based testing platform, termed “CRISPR-HBV,” for ultrasensitive, highly specific, and rapid detection of two major HBV genotypes (HBV-B and HBV-C) in clinical application. The CRISPR-HBV employed multiple cross displacement amplification (MCDA) for rapid preamplification and then Cas12b–based detection for decoding the targets. Finally, the detection result was read out with real-time fluorescence and a lateral flow biosensor. The sensitivity of CRISPR-HBV was 10 copies per test. The specificity was one hundred percent, and no cross reactions were observed in other HBV genotypes and pathogens. The whole detection process, including DNA template extraction (15 min), preamplification reaction of MCDA (30 min at 65°C), CRISPR-Cas12b–based detection (5 min at 37°C), and results readout (∼2 min), could be completed within 1 h. The feasibility of the CRISPR-HBV assay for genotyping HBV-B and -C as successfully validated with clinical samples. Hence, the CRISPR-HBV assay has remarkable potential to develop a point-of-care testing for identifying and distinguishing HBV genotypes B and C in clinical settings, especially in resource-scarcity countries.

PREVALENCE OF DIFFERENT HEPATITIS B VIRUS GENOTYPES AND RISK FACTORS ASSOCIATED AMONG SELECTED YEMENI PATIENTS WITH CHRONIC HEPATITIS B INFECTION

Background and aims: Hepatitis B virus infection is a significant public health crisis global. Hepatitis B virus genotyping is an important tool in epidemiological studies to determine the category and extent of treatment and to predict the outcome of chronic infections, for instance hepatocellular carcinoma and cirrhosis. The study designed to determine the prevalence of hepatitis B virus genotypes among Yemeni patients with chronic hepatitis B (CHB) and to evaluate some of the associated risk factors. Methods: Fifty patients (38 males, 12 females) with chronic hepatitis B from Al-Thawra Modern General Hospital, Al-Kuwait University Hospital, and AL-Gomhoria Hospital were included. HBV DNA was first detected by conventional PCR then HBV genotypes were determined using nested and multiplex PCR. Results: Mixed HBV genotypes (A+B+C+D+E), (A+B+C+D+E+F), and (A+B+C+D) were found to be the most prevalent (60 %), it is followed by genotype D (16 %), genotype B (16%) and genotype A (8%), whereas C, E, and F genotype were not found individually among the study population. Blood transfusion was associated with mixed infection (χ2=13.06; p= 0.005). Conclusions: In assumption, this study demonstrates the general prevalence of hepatitis B virus genotypes among HBV-infected Yemeni hepatitis B patients who request medical consideration in a hospital. In mono-genotype HBV infection, genotype B and D were the most prevalent genotypes. In HBV mixed genotype infection, the A/B/C/D/E genotype was the most prevalent in the study area. In the future, based on genotype, clinical trials and treatment regimens must be individually assumed to efficiently manage chronic HBV infection. To this end, a prospective nationwide population study of HBV genotype spreading and clinical outcomes is suggested. Peer Review History: Received: 15 May 2021; Revised: 11 June; Accepted: 27 June, Available online: 15 July 2021 Academic Editor: Ahmad Najib, Universitas Muslim Indonesia, Makassar, Indonesia, [email protected] UJPR follows the most transparent and toughest ‘Advanced OPEN peer review’ system. The identity of the authors and, reviewers will be known to each other. This transparent process will help to eradicate any possible malicious/purposeful interference by any person (publishing staff, reviewer, editor, author, etc) during peer review. As a result of this unique system, all reviewers will get their due recognition and respect, once their names are published in the papers. We expect that, by publishing peer review reports with published papers, will be helpful to many authors for drafting their article according to the specifications. Auhors will remove any error of their article and they will improve their article(s) according to the previous reports displayed with published article(s). The main purpose of it is ‘to improve the quality of a candidate manuscript’. Our reviewers check the ‘strength and weakness of a manuscript honestly’. There will increase in the perfection, and transparency. Received file: Reviewer's Comments: Average Peer review marks at initial stage: 6.5/10 Average Peer review marks at publication stage: 7.5/10 Reviewer(s) detail: Dr. Salfarina Ramli, Department of Pharmacology and Pharmaceutical Chemistry, Faculty of Pharmacy, Universiti Teknologi MARA, 42300 Puncak Alam, Selangor, Malaysia. [email protected] Dr. Asia Selman Abdullah, University of Basrah, Iraq, [email protected] Similar Articles: EXPLOSION OF HEPATITIS B AND C VIRUSES AMONG HEMODIALYSIS PATIENTS AS A RESULT OF HEMODIALYSIS CRISIS IN YEMEN PREVALENCE AND GENOTYPING OF HEPATITIS C VIRUS IN HEMODIALYSIS PATIENTS AND EVALUATION OF HCV-CORE ANTIGEN TEST IN SCREENING PATIENTS FOR DIALYSIS IN SANA'A CITY, YEMEN PREVALENCE OF HBV AND HCV; AND THEIR ASSOCIATED RISK FACTORS AMONG PUBLIC HEALTH CENTER CLEANERS AT SELECTED PUBLIC HEALTH CENTERS IN SANA'A CITY-YEMEN

Distribution of Hepatitis B Genotypes Among Hepatitis B Patients Visiting Decode Genomics Research Center

The hepatitis B virus (HBV) is non-cytopathic, hepatotropic and enveloped virus which causes Hepatitis B and infects the liver causing inflammation and hepatocellular necrosis. The genome sequence database shows that HBV has ten genetic diversities (A-J) ten in which the HBV genotypes, I and J are the new one. Three major genotypes of HBV (A, C and D) were found in Nepal. Despite being a low prevalence, Nepal has a diversity of hepatitis B. Hence, the study aims to determine the distribution of hepatitis B genotypes among the hepatitis B patients visiting Decode Genomics Research Center. HBV genotypes were determined by using a simpler, more rapid, and more specific genotyping system for HBV involving PCR using type-specific primers. Our study showed that different HBV genotypes were identified in which genotype D to be predominant one followed by C and also showed presence of genotype A, B and F. Many recombinant genotypes were also present in our study.

Prevalence and Genetic Diversity of HAV and HBV Viruses among Jaundice Patients at Coast General Hospital, Mombasa County, Kenya

Background: Hepatitis A and B causes morbidity and mortality among patients. This study determined the proportion of hepatitis A, B viruses (HAV, HBV) and genetic diversity of HBV among jaundice patients at the Coast General Hospital, Mombasa County, Kenya. Methods: A cross-sectional study was conducted among 222 patients; recruited and screened for hepatitis B surface antigen (HBsAg) and anti-HAV IgM. Viral deoxyribonucleic acid (DNA) was extracted from positive samples; partial hepatitis B virus-pol (HBV-pol) gene amplified, directly sequenced and generated sequences phylogenetically analysed using MEGA X software. Demographic characteristics were compared in relation to HBV infection using Chi-square. Results: Forty-seven (21.2%) out of the 222 patients tested positive for HBV while no HAV was detected. Among those infected, n = 8 (3.6%) were females and n = 39 (17.6%) males. Forty-five samples amplified and sequenced successfully. However, two samples failed to amplify. Phylogenetic analysis revealed HBV A1 genotype [n = 35 (74.5%)] was most predominant. A3, B and C2 genotypes each occurred [n = 1 (0.02%)]. This study revealed co-existence of HBV A3, B and C2 genotypes that have not yet been detected in this region. Conclusion: HBV A1 genotype remains the predominant genotypes in this region. The detected HBV prevalence indicates possible high transmission with possibility of increasing trends of HBV genotypes based on revelation of existence of new genotypes in this region.