Genetic diversity in enhancer II region of HBV genotype D and its association with advanced liver diseases

Background Hepatitis B Virus (HBV) is one of the most common human infectious agents, and the mutations in its genome may cause chronic hepatitis (CH), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). This study was designed to characterize the enhancer-II (Enh-II) region of X gene in HBV positive patients to assess the association of such mutations with CH, LC, and HCC. Methods HBV positive samples (N = 200) with patients’ demographic and clinical data were collected from different regions of Khyber Pakhtunkhwa (KP), Pakistan. The Enh-II region of the HBx gene was sequenced and zanalyzed for polymorphism associated with advanced liver disease. Univariate and logistic regression analyses were performed to evaluate potent mutations associated with a risk for LC and HCC. Results HBV Enh-II region sequences analysis revealed 25 different mutations. The highest frequency of mutations S101F (62.2%), A102V/R/G/I (56.25%), M103L/A (68.75%)were found in HCC, followed in LC and CH patients as 57.1%, 42.8%, 28.52% 16%, 15.2% and 18.4% respectively. H94 deletion in the α-box of the Enh-II region, associated with a high risk of HCC was found in half of the HCC patients. This deletion was present in 28.5% of LC and 6.5% of CH patients. Importantly, the high frequency of some notable mutations such as E109A/Y, A110S/K, Y111D/E, and F112L was first time reported in the entire study population. The frequencies of these mutations were high in HCC (43.75%, 37.5%, 50% and 43.75% respectively) as compared to LC (14.28%, 14.28%, 28.2% and 42.8%) and CH patients (12.8%, 15.2%, 16.8% and 16% respectively). Conclusion Mutations associated with LC and HCC are prevalent in the Enh-II region in Pakistani HBV isolates. The mutations found are alarming in CH patients as these may progress to LC and HCC in a large number of patients.

Morphological and Molecular Characterization of Trichuris sp. (Nematoda: Trichuridae) in Crested Porcupines (Hystrix cristata; Rodentia: Hystricidae) from Italy

Adult specimens of Trichuris sp. collected from crested porcupines (Hystrix cristata) from Italy were characterized using an integrative taxonomic approach involving morphological and molecular tools. The morphological features of this Trichuris sp. were compared to data already available for Trichuris spp. from Hystrix sp., revealing diagnostic traits, such as spicule length in males or vulva shape in females, which distinguish this Trichuris sp. from the other species. Evidence from sequences analysis of the partial mitochondrial COX1 region indicated that the taxon under study is a distinct lineage. Biometrical and genetic data suggested this Trichuris sp. to be a valid and separated taxon. However, since molecular data from other Trichuris spp. infecting Hystrix, such as T. infundibulus, T. hystricis, T. javanica, T.landak and T. lenkorani, are missing in public repositories, the number and identity of distinct lineages able to infect porcupines remain only partially defined.

Sequences analysis of chloroplast psbA-trnH region in Citrus L. (Rutaceae) species from the Aegean region of Turkey

In this study, sequences analysis of some Citrus species distributed in Turkey's Aegean region was based on the cpDNA psbA-trnH region. The sequences for psbA-trnH regions of the outgroups were retrieved from NCBI GenBank. Genomic DNA was isolated from healthy and green leaves. Total genomic DNA was extracted using GeneMark DNA isolation Plant Kit. The psbA-trnH region was amplified using primers psbA and trnH. DNA sequences were edited using the Sequencher 5.4.6. Sequencing data were analyzed using MEGA 6.0 software. Maximum likelihood (ML) tree was created to determine the relationships between Citrus taxa. cpDNA psbA-trnH sequences ranged between 426 and 470 nucleotides. Maximum likelihood phylogenetic tree is composed of two clades. The divergence values differed between 0.000 and 0.012. According to the results of the study, the separation of Citrus species in phylogenetic tree obtained with psbA-trnH sequence data was realized. However, it has been found that cpDNA psbA-trnH sequence populations of species belong together. In addition, the phylogenetic relationship between the sequence data of some species belonging to the Rutaceae family taken from NCBI and Citrus species was revealed.

Sequence Analysis of the Internal Transcribed Spacer (ITS) Region of the Nuclear Ribosomal DNA (nrDNA), and trnL Intron of Chloroplast DNA (cpDNA) of the Chrysochamela (Fenzl) Boiss. (Brassicaceae) In Turkey

Brassicaceae family is an important one since it includes many economic and significant industrial oilseeds, spices, vegetables and some forage plants. In this study, sequences analysis among Chrysochamela (Brassicaceae) species distributed in Turkey were conducted nrDNA ITS and cpDNA trnL intron sequences. Chrysochamela species were collected and brought to the laboratory. ITS and trnL intron sequences were corrected with BioEdit and FinchTV programs. As a result of the study, ITS nucleotide compound compositions were determined as 22.7% T, 29.1 C, 21.5% A and 26.7% G. The lowest distance was 0.000 and the highest distance was 0.037. The phylogenetic tree obtained using the MEGA 6.0 program consists of two large groups. According to trnL intron sequences 37.9% T, 18.4 C, 28.3% A and 15.5% G. Nucleotide compound compositions were determined. The genetic distance between species was determined between 0.000 and 0.022. Maximum likelihood phylogenetic tree consists of two large groups. As a result, phylogenetic analyzes using ITS and trnL intron sequences were compatible with each other. It was also in past studies found to be supported by morphological, anatomical and RAPD data.

Nanopore sequencing of SARS-CoV-2: Comparison of short and long PCR-tiling amplicon protocols

Surveillance of the SARS-CoV-2 variants including the quickly spreading mutants by rapid and near real-time sequencing of the viral genome provides an important tool for effective health policy decision making in the ongoing COVID-19 pandemic. Here we evaluated PCR-tiling of short (~400-bp) and long (~2 and ~2.5-kb) amplicons combined with nanopore sequencing on a MinION device for analysis of the SARS-CoV-2 genome sequences. Analysis of several sequencing runs demonstrated that using the long amplicon schemes outperforms the original protocol based on the 400-bp amplicons. It also illustrated common artefacts and problems associated with PCR-tiling approach, such as uneven genome coverage, variable fraction of discarded sequencing reads, including human and bacterial contamination, as well as the presence of reads derived from the viral sub-genomic RNAs.

The presence of Ehrlichia canis in Rhipicephalus bursa ticks collected from ungulates in continental Eastern Europe

Introduction Rhipicephalus bursa is a common tick parasite of small-to-medium size ungulates, principally in warm, temperate, and subtropical areas. Although common in livestock and showing a wide geographic distribution, its epidemiological role in tick-borne bacterial disease is barely known. This study addressed the knowledge gap and aimed to screen for the presence of Anaplasmataceae and spotted fever group (SFG) Rickettsia species in R. bursa ticks collected from domestic animals in Romania, Eastern Europe. Material and Methods A total of 64 pools of R. bursa ticks collected from small ungulates were tested by PCR for Anaplasmataceae DNA presence using group-specific primers. Specific testing was performed for Anaplasma marginale/A. centrale/A. ovis, A. platys, A. phagocytophilum, Ehrlichia canis, and SFG Rickettsia. The positive samples were purified and sequenced, and sequences analysis was used to identify the species and to confirm the PCR results. Results The only pathogen identified in this study was E. canis. The obtained sequences confirmed the PCR results. The presence of E. canis in R. bursa in Romania and in ticks from sheep was shown for the first time in this study. Conclusion Based on these findings, it may be presumed that the E. canis DNA originated from ticks; however, the vectorial role of R. bursa (and other arthropod species) in the transmission of E. canis should be proved experimentally.

Molecular Identification of Cryptosporidium viatorum Infection in a Patient Suffering from Unusual Cryptosporidiosis in West Bengal, India

In this study, we have collected and screened a total of 268 stool samples from diarrheal patients admitted to an Infectious disease hospital in Kolkata for the presence of Cryptosporidium spp. The initial diagnosis was carried out by microscopy followed by genus specific polymerase chain reaction assays based on 70 kDa heat shock proteins (HSP70). DNA sequencing of the amplified locus has been employed for determination of genetic diversity of the local isolates. Out of 268 collected samples, 12 (4.48%) were positive for Cryptosporidium spp. Sequences analysis of 70 kDa heat shock proteins locus in 12 Cryptosporidium local isolates revealed that 2.24% and 1.86% of samples were showing 99% to 100% identity with C. parvum and C. hominis. Along with the other 2 major species one recently described globally distributed pathogenic species Cryptosporidium viatorum has been identified. The HSP70 locus sequence of the isolate showed 100% similarity with a previously described isolate of C. viatorum (Accession No. JX978274.1, JX978273.1, and JN846706.1) present in GenBank.

Screening of Some Romanian Raw Honeys and Their Probiotic Potential Evaluation

This study aimed to characterize raw honeys from different geographical origins in Romania, in respect of chemical composition, microbiological examination and evaluate their probiotic potential. The physico-chemical determinations were performed in APHIS-DIA Laboratory, Cluj-Napoca, Romania, using standard validated methods. Bacterial identification was performed for each sample and each colony type using Vitek® 2 Compact 15 system and PCR amplification using 16S rDNA bacterial universal primers (27F, 1492R), species being confirm by sequences analysis. In five raw honey samples, we have identified probiotic bacteria, such as: Bacillus mycoides, Bacillus thuringiensis, Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus velezensis. Generally, all honey samples meet the standard values for chemical composition. However, one sample having 7.44% sucrose was found to have also probiotics bacteria from the genus Bacillus because sucrose is a substrate for probiotics development. In conclusion, the Romanian raw honey can be a potential reservoir of probiotics, which confer a health benefit for consumers.

Nanopore Sequencing of SARS-CoV-2: Comparison of Short and Long PCR-tiling Amplicon Protocols

Surveillance of the SARS-CoV-2 variants including the quickly spreading mutants by rapid and near real-time sequencing of the viral genome provides an important tool for effective health policy decision making in the ongoing COVID-19 pandemic. Here we evaluated PCR-tiling of short (~400-bp) and long (~2 and ~2.5-kb) amplicons combined with nanopore sequencing on a MinION device for analysis of the SARS-CoV-2 genome sequences. Analysis of several sequencing runs demonstrated that using the long amplicon schemes outperforms the original protocol based on the 400-bp amplicons. It also illustrated common artefacts and problems associated with this approach, such as uneven genome coverage, variable fraction of discarded sequencing reads, as well as the reads derived from the viral sub-genomic RNAs and/or human and bacterial contamination.