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Hepatitis E virus (HEV; family Hepeviridae, genus Orthohepevirus) (1) is an RNA virus that causes acute viral hepatitis in humans. Five HEV genotypes are known to infect humans. Genotypes 1 [...]

Our aim was to develop an accurate, highly sensitive method for HBV genotype determination and detection of genotype mixtures. We examined the preS and 5′ end of the HBV X gene (5X) regions of the HBV genome using next-generation sequencing (NGS). The 1852 haplotypes obtained were subjected to genotyping via the Distance-Based discrimination method (DB Rule) using two sets of 95 reference sequences of genotypes A–H. In clinical samples from 125 patients, the main genotypes were A, D, F and H in Caucasian, B and C in Asian and A and E in Sub-Saharan patients. Genotype mixtures were identified in 28 (22.40%) cases, and potential intergenotypic recombination was observed in 29 (23.20%) cases. Furthermore, we evaluated sequence conservation among haplotypes classified into genotypes A, C, D, and E by computing the information content. The preS haplotypes exhibited limited shared conserved regions, whereas the 5X haplotypes revealed two groups of conserved regions across the genotypes assessed. In conclusion, we developed an NGS-based HBV genotyping method utilizing the DB Rule for genotype classification. We identified two regions conserved across different genotypes at 5X, offering promising targets for RNA interference-based antiviral therapies. [ABSTRACT FROM AUTHOR]

In quasispecies diversity studies, the comparison of two samples of varying sizes is a common necessity. However, the sensitivity of certain diversity indices to sample size variations poses a challenge. To address this issue, rarefaction emerges as a crucial tool, serving to normalize and create fairly comparable samples. This study emphasizes the imperative nature of sample size normalization in quasispecies diversity studies using next-generation sequencing (NGS) data. We present a thorough examination of resampling schemes using various simple hypothetical cases of quasispecies showing different quasispecies structures in the sense of haplotype genomic composition, offering a comprehensive understanding of their implications in general cases. Despite the big numbers implied in this sort of study, often involving coverages exceeding 100,000 reads per sample and amplicon, the rarefaction process for normalization should be performed with repeated resampling without replacement, especially when rare haplotypes constitute a significant fraction of interest. However, it is noteworthy that different diversity indices exhibit distinct sensitivities to sample size. Consequently, some diversity indicators may be compared directly without normalization, or instead may be resampled safely with replacement. [ABSTRACT FROM AUTHOR]

The repeated failure to treat patients chronically infected with hepatitis E (HEV) and C (HCV) viruses, despite the absence of resistance-associated substitutions (RAS), particularly in response to prolonged treatments with the mutagenic agents of HEV, suggests that quasispecies structure may play a crucial role beyond single point mutations. Quasispecies structured in a flat-like manner (referred to as flat-like) are considered to possess high average fitness, occupy a significant fraction of the functional genetic space of the virus, and exhibit a high capacity to evade specific or mutagenic treatments. In this paper, we studied HEV and HCV samples using high-depth next-generation sequencing (NGS), with indices scoring the different properties describing flat-like quasispecies. The significance of these indices was demonstrated by comparing the values obtained from these samples with those from acute infections caused by respiratory viruses (betacoronaviruses, enterovirus, respiratory syncytial viruses, and metapneumovirus). Our results revealed that flat-like quasispecies in HEV and HCV chronic infections without RAS are characterized by numerous low-frequency haplotypes with no dominant one. Surprisingly, these low-frequency haplotypes (at the nucleotide level) exhibited a high level of synonymity, resulting in much lower diversity at the phenotypic level. Currently, clinical approaches for managing flat-like quasispecies are lacking. Here, we propose methods to identifying flat-like quasispecies, which represents an essential initial step towards exploring alternative treatment protocols for viruses resistant to conventional therapies. [ABSTRACT FROM AUTHOR]

Phage therapy is still in its infancy, but it is increasingly promising as a future alternative for treating antibiotic-resistant bacteria. To investigate the effect of phages on Mycobacterium abscessus complex (MABC), we isolated 113 environmental phages, grown them to high titres, and assayed them on MABC clinical strains through the spot test. Of all the phages, only 16 showed killing activity. Their activity was so temperate to MABC that they could not generate any plaque-forming units (PFUs). The Appelmans method of directed evolution was carried out to evolve these 16 phages into more lytic ones. After only 11 of 30 rounds of evolution, every single clinical strain in our collection, including those that were unsusceptible up to this point, could be lysed by at least one phage. The evolved phages were able to form PFUs on the clinical strains tested. Still, they are temperate at best and require further training. The genomes of one random parental phage and three random evolved phages from Round 13 were sequenced, revealing a diversity of clusters and genes of a variety of evolutionary origins, mostly of unknown function. These complete annotated genomes will be key for future molecular characterisations. [ABSTRACT FROM AUTHOR]

Here, we report the in-host hepatitis E virus (HEV) quasispecies evolution in a chronically infected patient who was treated with three different regimens of ribavirin (RBV) for nearly 6 years. Sequential plasma samples were collected at different time points and subjected to RNA extraction and deep sequencing using the MiSeq Illumina platforms. Specifically, we RT-PCR amplified a single amplicon from the core region located in the open-reading frame 2 (ORF2). At the nucleotide level (genotype), our analysis showed an increase in the number of rare haplotypes and a drastic reduction in the frequency of the master (most represented) sequence during the period when the virus was found to be insensitive to RBV treatment. Contrarily, at the amino acid level (phenotype), our study revealed conservation of the amino acids, which is represented by a high prevalence of the master sequence. Our findings suggest that using mutagenic antivirals concomitant with high viral loads can lead to the selection and proliferation of a rich set of synonymous haplotypes that express the same phenotype. This can also lead to the selection and proliferation of conservative substitutions that express fitness-enhanced phenotypes. These results have important clinical implications, as they suggest that using mutagenic agents as a monotherapy treatment regimen in the absence of sufficiently effective viral inhibitors can result in diversification and proliferation of a highly diverse quasispecies resistant to further treatment. Therefore, such approaches should be avoided whenever possible. [ABSTRACT FROM AUTHOR]

Shortly after the beginning of the SARS-CoV-2 pandemic, many countries implemented sewage sentinel systems to monitor the circulation of the virus in the population. A fundamental part of these surveillance programs is the variant tracking through sequencing approaches to monitor and identify new variants or mutations that may be of importance. Two of the main sequencing platforms are Illumina and Oxford Nanopore Technologies. Here, we compare the performance of MiSeq (Illumina) and MinION (Oxford Nanopore Technologies), as well as two different data processing pipelines, to determine the effect they may have on the results. MiSeq showed higher sequencing coverage, lower error rate, and better capacity to detect and accurately estimate variant abundances than MinION R9.4.1 flow cell data. The use of different variant callers (LoFreq and iVar) and approaches to calculate the variant proportions had a remarkable impact on the results generated from wastewater samples. Freyja, coupled with iVar, may be more sensitive and accurate than LoFreq, especially with MinION data, but it comes at the cost of having a higher error rate. The analysis of MinION R10.4.1 flow cell data using Freyja combined with iVar narrows the gap with MiSeq performance in terms of read quality, accuracy, sensitivity, and number of detected mutations. Although MiSeq should still be considered as the standard method for SARS-CoV-2 variant tracking, MinION's versatility and rapid turnaround time may represent a clear advantage during the ongoing pandemic. [ABSTRACT FROM AUTHOR]

Hepatitis is an inflammation of the liver whose etiology is very heterogeneous. The most common cause of hepatitis is viral infections from hepatotropic viruses, including hepatitis A, B, C, D and E. However, other factors such as infections from other agents, metabolic disorders, or autoimmune reactions can also contribute to hepatitis, albeit to a lesser extent. On April 5, 2022, the United Kingdom Health Security Agency alerted the World Health Organization (WHO) on the increased incidence of severe acute hepatitis of unknown causes (not AE) in previously healthy young children, with symptoms of liver failure that in some cases required liver transplantation. By July 2022, 1,296 cases were reported in 37 countries. Acute hepatitis of unknown causes is not an exceptional phenomenon: in fact, it represents more than 30% of cases of acute hepatitis in children, however in the present instance the large proportion of severe cases was surprising and alarming (6% of liver transplants and almost 3% mortality). Multiple hypotheses have been proposed to explain the etiology of such higher proportion of acute hepatitis, including their co-occurrence in the context of COVID-19 pandemic. This is a review of the history of a clinical threat that has put in check a world health care system highly sensitized by the current COVID-19 pandemics, and that it looks like has ended with the arguments that the severe acute pediatric hepatitis is caused by Adeno-associated virus 2 (AAV2) infection associated with a coinfection with a helper virus (human Adenovirus HAdV or human herpesvirus 6) in susceptible children carrying HLA-class II antigen HLA-DRB1*04:01. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) are potential diagnostic and prognostic biomarkers. However, discrepancies in miRNA patterns and their validation are still frequent due to differences in sample origin, EV isolation, and miRNA sequencing methods. The aim of the present study is to find a reliable EV isolation method for miRNA sequencing, adequate for clinical application. To this aim, two comparative studies were performed in parallel with the same human plasma sample: (i) isolation and characterization of EVs obtained using three procedures: size exclusion chromatography (SEC), iodixanol gradient (GRAD), and its combination (SEC+GRAD) and (ii) evaluation of the yield of miRNA sequences obtained using NextSeq 500 (Illumina) and three miRNA library preparation protocols: NEBNext, NEXTFlex, and SMARTer smRNA-seq. The conclusion of comparison (i) is that recovery of the largest amount of EVs and reproducibility were attained with SEC, but GRAD and SEC+GRAD yielded purer EV preparations. The conclusion of (ii) is that the NEBNext library showed the highest reproducibility in the number of miRNAs recovered and the highest diversity of miRNAs. These results render the combination of GRAD EV isolation and NEBNext library preparation for miRNA retrieval as adequate for clinical applications using plasma samples. [ABSTRACT FROM AUTHOR]

Despite being minority languages like many others, sign languages have traditionally remained absent from the agendas of policy makers and language planning and policies. In the past two decades, though, this situation has started to change at different paces and to different degrees in several countries. In this article, the author describes the highlights of the process that led to the legal recognition of Catalan Sign Language (Llengua de signes catalana, LSC) in the context of Catalonia and Spain, and he analyzes the parallelisms and the differences in both processes. Finally, the author reviews the new perspectives and the challenges that have arisen as a consequence of these processes. (Contains 9 notes.)

This paper investigates whether the semantic and phonological levels in speech production are specific to spoken languages or universal across modalities. We examined semantic and phonological effects during Catalan Signed Language (LSC: Llengua de Signes Catalana) production using an adaptation of the picture-word interference task: native and non-native signers were asked to sign picture names while ignoring signs produced in the background. The results showed semantic interference effects for semantically related distractor signs and phonological facilitation effects when target signs and distractor signs shared either "Handshape" or "Movement" but phonological interference effects when target and distractor shared "Location". The results suggest that the general distinction between semantic and phonological levels seems to hold across modalities. However, differences in sign language and spoken production become evident in the mechanisms underlying phonological encoding, shown by the different role that "Location", "Handshape", and "Movement" play during phonological encoding in sign language. (Contains 2 tables and 1 figure.)

Epidemics and pandemics have occurred since the beginning of time, resulting in millions of deaths. Many such disease outbreaks are caused by viruses. Some viruses, particularly RNA viruses, are characterized by their high genetic variability, and this can affect certain phenotypic features: tropism, antigenicity, and susceptibility to antiviral drugs, vaccines, and the host immune response. The best strategy to face the emergence of new infectious genomes is prompt identification. However, currently available diagnostic tests are often limited for detecting new agents. High-throughput next-generation sequencing technologies based on metagenomics may be the solution to detect new infectious genomes and properly diagnose certain diseases. Metagenomic techniques enable the identification and characterization of disease-causing agents, but they require a large amount of genetic material and involve complex bioinformatic analyses. A wide variety of analytical tools can be used in the quality control and pre-processing of metagenomic data, filtering of untargeted sequences, assembly and quality control of reads, and taxonomic profiling of sequences to identify new viruses and ones that have been sequenced and uploaded to dedicated databases. Although there have been huge advances in the field of metagenomics, there is still a lack of consensus about which of the various approaches should be used for specific data analysis tasks. In this review, we provide some background on the study of viral infections, describe the contribution of metagenomics to this field, and place special emphasis on the bioinformatic tools (with their capabilities and limitations) available for use in metagenomic analyses of viral pathogens. [ABSTRACT FROM AUTHOR]

What takes decades, centuries or millennia to happen with a natural ecosystem, it takes only days, weeks or months with a replicating viral quasispecies in a host, especially when under treatment. Some methods to quantify the evolution of a quasispecies are introduced and discussed, along with simple simulated examples to help in the interpretation and understanding of the results. The proposed methods treat the molecules in a quasispecies as individuals of competing species in an ecosystem, where the haplotypes are the competing species, and the ecosystem is the quasispecies in a host, and the evolution of the system is quantified by monitoring changes in haplotype frequencies. The correlation between the proposed indices is also discussed, and the R code used to generate the simulations, the data and the plots is provided. The virtues of the proposed indices are finally shown on a clinical case. [ABSTRACT FROM AUTHOR]

The changes occurring in viral quasispecies populations during infection have been monitored using diversity indices, nucleotide diversity, and several other indices to summarize the quasispecies structure in a single value. In this study, we present a method to partition quasispecies haplotypes into four fractions according to their fitness: the master haplotype, rare haplotypes at two levels (those present at <0.1%, and those at 0.1–1%), and a fourth fraction that we term emerging haplotypes, present at frequencies >1%, but less than that of the master haplotype. We propose that by determining the changes occurring in the volume of the four quasispecies fitness fractions together with those of the Hill number profile we will be able to visualize and analyze the molecular changes in the composition of a quasispecies with time. To develop this concept, we used three data sets: a technical clone of the complete SARS-CoV-2 spike gene, a subset of data previously used in a study of rare haplotypes, and data from a clinical follow-up study of a patient chronically infected with HEV and treated with ribavirin. The viral response to ribavirin mutagenic treatment was selection of a rich set of synonymous haplotypes. The mutation spectrum was very complex at the nucleotide level, but at the protein (phenotypic/functional) level the pattern differed, showing a highly prevalent master phenotype. We discuss the putative implications of this observation in relation to mutagenic antiviral treatment. [ABSTRACT FROM AUTHOR]

Background: Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the main concern is whether reinfections are possible, and which are the associated risk factors. This study aims to describe the clinical and molecular characteristics of 24 sequence-confirmed reinfection SARS-CoV-2 cases over 1 year in Barcelona (Catalonia, Spain). Methods: Patients with > 45 days between two positive PCR tests regardless of symptoms and negative tests between episodes were initially considered as suspected reinfection cases from November 2020 to May 2021. Whole-genome sequencing (WGS) was performed to confirm genetic differences between consensus sequences and for phylogenetic studies based on PANGOLIN nomenclature. Reinfections were confirmed by the number of mutations, change in lineage, or epidemiological criteria. Results: From 39 reported suspected reinfection cases, complete viral genomes could be sequenced from both episodes of 24 patients, all were confirmed as true reinfections. With a median age of 44 years (interquartile range [IQR] 32–65), 66% were women and 58% were healthcare workers (HCWs). The median days between episodes were 122 (IQR 72–199), occurring one-third within 3 months. Reinfection episodes were frequently asymptomatic and less severe than primary infections. The absence of seroconversion was associated with symptomatic reinfections. Only one case was reinfected with a variant of concern (VOC). Conclusion: Severe acute respiratory syndrome coronavirus 2 reinfections can occur in a shorter time than previously reported and are mainly found in immunocompetent patients. Surveillance through WGS is useful to identify viral mutations associated with immune evasion. [ABSTRACT FROM AUTHOR]

Simple Summary: In this unicentric prospective study, we analyzed the most prevalent mutations in HCC (TERT promoter, TP53, CTNNB1, AXIN1 and ARID1A) in plasma cfDNA by next-generation sequencing, aiming to elucidate their value as prognostic noninvasive biomarkers. Total cfDNA (cut-off value 2 ng/µL), number of mutated genes and number of detectable mutations on cfDNA were significantly related to mortality. Number of mutated genes and number of detected mutations in cfDNA and the ratio between number of mutations and total amount of cfDNA were also significantly associated with recurrence. Detection of more than four mutations in cfDNA correlated with a higher risk of death. Dynamic changes in cfDNA mutations were detected prior to radiological detection of HCC recurrence. We believe that these results support the proof of principle and launching of validation studies to confirm that total cfDNA and detection of prevalent HCC mutations could have prognostic implications in early-stage HCC patients. Background: Cell-free DNA (cfDNA) concentrations have been described to be inversely correlated with prognosis in cancer. Mutations in HCC-associated driver genes in cfDNA have been reported, but their relation with patient's outcome has not been described. Our aim was to elucidate whether mutations found in cfDNA could be representative from those present in HCC tissue, providing the rationale to use the cfDNA to monitor HCC. Methods: Tumoral tissue, paired nontumor adjacent tissue and blood samples were collected from 30 HCC patients undergoing curative therapies. Deep sequencing targeting HCC driver genes was performed. Results: Patients with more than 2 ng/µL of cfDNA at diagnosis had higher mortality (mean OS 24.6 vs. 31.87 months, p = 0.01) (AUC = 0.782). Subjects who died during follow-up, had a significantly higher number of mutated genes (p = 0.015) and number of mutations (p = 0.015) on cfDNA. Number of mutated genes (p = 0.001), detected mutations (p = 0.001) in cfDNA and ratio (number of mutations/cfDNA) (p = 0.003) were significantly associated with recurrence. However, patients with a ratio (number of mutations/cfDNA) above 6 (long-rank p = 0.0003) presented a higher risk of recurrence than those with a ratio under 6. Detection of more than four mutations in cfDNA correlated with higher risk of death (long-rank p = 0.042). Conclusions: In summary, cfDNA and detection of prevalent HCC mutations could have prognostic implications in early-stage HCC patients [ABSTRACT FROM AUTHOR]

Background: Hepatitis C virus (HCV) is a major cause of chronic liver infection with 71 million people infected worldwide. Pakistan has the second highest prevalence of HCV infection and more than half (52%) of Pakistani living in Spain reside in Barcelona. The aim of this study was to analyse the seroprevalence and viraemic rate and determine the genotypes and subtypes of HCV among Pakistanis living in the southern metropolitan area of Barcelona. Methods: We included all Pakistani patients seeking primary healthcare in the southern metropolitan area of Barcelona from August 2011 to July 2014. Serum samples were screened for HCV antibodies. HCV viral load was determined by reverse transcription polymerase chain reaction and genotypes and subtypes were performed using Versant HCV Genotype and/or deep-sequencing. Screening for hepatitis B virus (HBV) was also carried out. Results: Among 5877 Pakistani patients, 565 (9.61%) were screened for anti-HCV antibodies, with 68 (12.04%) being positive. The viral load was determined in 65, with 31 presenting active infection and the viraemic rate was 47.69% (95% confidence interval 36.02– 59.62). HCV genotyping and subtyping were performed in 24 individuals. Most infections corresponded to HCV genotype 3 (91.67%), and high resolution HCV subtyping was performed in 18 samples, 16 of which presented subtype 3a. One subject presented HBV coinfection with undetectable HBV DNA. During the study period, we identified a possible case of HCV vertical transmission followed by spontaneous viraemia clearance in a chronically infected mother with a C/T IL28B genetic polymorphism. Conclusion: These results suggest that general HCV screening protocols in patients from high prevalence countries, such as Pakistan, would be helpful to identify and treat active HCV infections. This could avoid further transmission and contribute to building targeted health policies for micro-elimination of HCV infection in specific communities. [ABSTRACT FROM AUTHOR]

Deletions in the 3′ end region of the hepatitis B virus (HBV) X open reading frame (HBX) may affect the core promoter (Cp) and have been frequently associated with hepatocellular carcinoma (HCC). The aim of this study was to investigate the presence of variants with deletions and/or insertions (Indels) in this region in the quasispecies of 50 chronic hepatitis B (CHB) patients without HCC. We identified 103 different Indels in 47 (94%) patients, in a median of 3.4% of their reads (IQR, 1.3–8.4%), and 25% (IQR, 13.1–40.7%) of unique sequences identified in each quasispecies (haplotypes). Of those Indels, 101 (98.1%) caused 44 different altered stop codons, the most commonly observed were at positions 128, 129, 135, and 362 (putative position). Moreover, 39 (37.9%) Indels altered the TATA-like box (TA) sequences of Cp; the most commonly observed caused TA2 + TA3 fusion, creating a new putative canonical TATA box. Four (8%) patients developed negative clinical outcomes after a median follow-up of 9.4 (8.7–12) years. In conclusion, we observed variants with Indels in the HBX 3′ end in the vast majority of our CHB patients, some of them encoding alternative versions of HBx with potential functional roles, and/or alterations in the regulation of transcription. [ABSTRACT FROM AUTHOR]