Genotyping Hepatitis B virus by Next-Generation Sequencing: Detection of Mixed Infections and Analysis of Sequence Conservation.

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.

In-Host Flat-like Quasispecies: Characterization Methods and Clinical Implications.

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.

Virus Quasispecies Rarefaction: Subsampling with or without Replacement?

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.

Comparison of Nanopore and Synthesis-Based Next-Generation Sequencing Platforms for SARS-CoV-2 Variant Monitoring in Wastewater.

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.

In-Host HEV Quasispecies Evolution Shows the Limits of Mutagenic Antiviral Treatments.

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.

Fitness-Dependent, Mild Mutagenic Activity of Sofosbuvir for Hepatitis C Virus.

The concept of a mild mutagen was coined to describe a minor mutagenic activity exhibited by some nucleoside analogues that potentiated their efficacy as antiretroviral agents. In the present study, we report the mild mutagen activity of sofosbuvir (SOF) for hepatitis C virus (HCV). Serial passages of HCV in human hepatoma cells, in the presence of SOF at a concentration well below its cytotoxic concentration 50 (CC50) led to pre-extinction populations whose mutant spectra exhibited a significant increase of C→U transitions, relative to populations passaged in the absence of SOF. This was reflected in an increase in several diversity indices that were used to characterize viral quasispecies. The mild mutagenic activity of SOF was largely absent when it was tested with isogenic HCV populations that displayed high replicative fitness. Thus, SOF can act as a mild mutagen for HCV, depending on HCV fitness. Possible mechanisms by which the SOF mutagenic activity may contribute to its antiviral efficacy are discussed.

Bioinformatic Tools for NGS-Based Metagenomics to Improve the Clinical Diagnosis of Emerging, Re-Emerging and New Viruses.

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.

Quantifying In-Host Quasispecies Evolution.

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.

msmsEDA & msmsTests: Label-Free Differential Expression by Spectral Counts.

msmsTests is an R/Bioconductor package providing functions for statistical tests in label-free LC-MS/MS data by spectral counts. These functions aim at discovering differentially expressed proteins between two biological conditions. Three tests are available: Poisson GLM regression, quasi-likelihood GLM regression, and the negative binomial of the edgeR package. The three models admit blocking factors to control for nuisance variables. To assure a good level of reproducibility a post-test filter is available, where (1) a minimum effect size considered biologically relevant, and (2) a minimum expression of the most abundant condition, may be set. A companion package, msmsEDA, proposes functions to explore datasets based on msms spectral counts. The provided graphics help in identifying outliers, the presence of eventual batch factors, and check the effects of different normalizing strategies. This protocol illustrates the use of both packages on two examples: A purely spike-in experiment of 48 human proteins in a standard yeast cell lysate; and a cancer cell-line secretome dataset requiring a biological normalization.

Quasispecies Fitness Partition to Characterize the Molecular Status of a Viral Population. Negative Effect of Early Ribavirin Discontinuation in a Chronically Infected HEV Patient.

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.

The frequency of defective genomes in Omicron differs from that of the Alpha, Beta and Delta variants.

The SARS-CoV-2 Omicron variant emerged showing higher transmissibility and possibly higher resistance to current COVID-19 vaccines than other variants dominating the global pandemic. In March 2020 we performed a study in clinical samples, where we found that a portion of genomes in the SARS-CoV-2 viral population accumulated deletions immediately before the S1/S2 cleavage site (furin-like cleavage site, PRRAR/S) of the spike gene, generating a frameshift and appearance of a premature stop codon. The main aim of this study was to determine the frequency of defective deletions in prevalent variants from the first to sixth pandemic waves in our setting and discuss whether the differences observed might support epidemiological proposals. The complete SARS-CoV-2 spike gene was deeply studied by next-generation sequencing using the MiSeq platform. More than 90 million reads were obtained from respiratory swab specimens of 78 COVID-19 patients with mild infection caused by the predominant variants circulating in the Barcelona city area during the six pandemic waves: B.1.5, B.1.1, B.1.177, Alpha, Beta, Delta, and Omicron. The frequency of defective genomes found in variants dominating the first and second waves was similar to that seen in Omicron, but differed from the frequencies seen in the Alpha, Beta and Delta variants. The changing pattern of mutations seen in the various SARS-CoV-2 variants driving the pandemic waves over time can affect viral transmission and immune escape. Here we discuss the putative biological effects of defective deletions naturally occurring before the S1/S2 cleavage site during adaption of the virus to human infection.

Ultra Deep Sequencing of Circulating Cell-Free DNA as a Potential Tool for Hepatocellular Carcinoma Management.

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.

Prevalence of Hepatitis C Virus Infection, Genotypes and Subtypes in Migrants from Pakistan in Barcelona, Spain.

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.

Hepatitis B Virus Variants with Multiple Insertions and/or Deletions in the X Open Reading Frame 3' End: Common Members of Viral Quasispecies in Chronic Hepatitis B Patients.

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.

Inspecting the Ribozyme Region of Hepatitis Delta Virus Genotype 1: Conservation and Variability.

The hepatitis delta virus (HDV) genome has an autocatalytic region called the ribozyme, which is essential for viral replication. The aim of this study was to use next-generation sequencing (NGS) to analyze the ribozyme quasispecies (QS) in order to study its evolution and identify highly conserved regions potentially suitable for a gene-silencing strategy. HDV RNA was extracted from 2 longitudinal samples of chronic HDV patients and the ribozyme (nucleotide, nt 688-771) was analyzed using NGS. QS conservation, variability and genetic distance were analyzed. Mutations were identified by aligning sequences with their specific genotype consensus. The main relevant mutations were tested in vitro. The ribozyme was conserved overall, with a hyper-conserved region between nt 715-745. No difference in QS was observed over time. The most variable region was between nt 739-769. Thirteen mutations were observed, with three showing a higher frequency: T23C, T69C and C64 deletion. This last strongly reduced HDV replication by more than 1 log in vitro. HDV Ribozyme QS was generally highly conserved and was maintained during follow-up. The most conserved portion may be a valuable target for a gene-silencing strategy. The presence of the C64 deletion may strongly impair viral replication, as it is a potential mechanism of viral persistence.

Dynamics of SARS-CoV-2 Alpha (B.1.1.7) variant spread: The wastewater surveillance approach.

Wastewater based epidemiology (WBE) offers an overview of the SARS-CoV-2 variants circulating among the population thereby serving as a proper surveillance method. The variant of concern (VOC) Alpha was first identified in September 2020 in the United Kingdom, and rapidly became dominant across Europe. Our objective was to elucidate the Alpha VOC outcompetition rate and identify mutations in the spike glycoprotein (S) gene, indicative of the circulation of the Alpha VOC and/or other variants in the population through wastewater analysis. In the period covered by this study (November 2020-April 2021), forteen wastewater treatment plants (WWTPs) were weekly sampled. The total number of SARS-CoV-2 genome copies per L (GC/L) was determined with a Real-Time qPCR, targeting the N gene. Surveillance of the Alpha VOC circulation was ascertained using a duplex RT-qPCR, targeting and discriminating the S gene. Our results showed that in a period of 6 weeks the Alpha VOC was present in all the studied WWTPs, and became dominant in 11 weeks on average. The outcompetition rates of the Alpha VOC were estimated, and their relationship with different parameters statistically analyzed. The rapid spread of the Alpha VOC was influenced by its initial input and by the previous circulation of SARS-COV-2 in the population. This latter point could be explained by its higher transmissibility, particularly advantadgeous when a certain degree of herd immunity exists. Moreover, the presence of signature mutations of SARS-COV-2 variants were established by deep-sequencing of the complete S gene. The circulation of the Alpha VOC in the area under study was confirmed, and additionally two combinations of mutations in the S glycoprotein (T73A and D253N, and S477N and A522S) that could affect antibody binding were identified.

Cross-sectional evaluation of circulating hepatitis B virus RNA and DNA: Different quasispecies?

BACKGROUND: Different forms of pregenomic and other hepatitis B virus (HBV) RNA have been detected in patients' sera. These circulating HBV-RNAs may be useful for monitoring covalently closed circular DNA activity, and predicting hepatitis B e-antigen seroconversion or viral rebound after nucleos(t)ide analog cessation. Data on serum HBV-RNA quasispecies, however, is scarce. It is therefore important to develop methodologies to thoroughly analyze this quasispecies, ensuring the elimination of any residual HBV-DNA. Studying circulating HBV-RNA quasispecies may facilitate achieving functional cure of HBV infection. AIM: To establish a next-generation sequencing (NGS) methodology for analyzing serum HBV-RNA and comparing it with DNA quasispecies. METHODS: Thirteen untreated chronic hepatitis B patients, showing different HBV-genotypes and degrees of severity of liver disease were enrolled in the study and a serum sample with HBV-DNA > 5 Log10 IU/mL and HBV-RNA > 4 Log10 copies/mL was taken from each patient. HBV-RNA was treated with DNAse I to remove any residual DNA, and the region between nucleotides (nt) 1255-1611 was amplified using a 3-nested polymerase chain reaction protocol, and analyzed with NGS. Variability/conservation and complexity was compared between HBV-DNA and RNA quasispecies. RESULTS: No HBV-DNA contamination was detected in cDNA samples from HBV-RNA quasispecies. HBV quasispecies complexity showed heterogeneous behavior among patients. The Rare Haplotype Load at 1% was greater in DNA than in RNA quasispecies, with no statistically significant differences (P = 0.1641). Regarding conservation, information content was equal in RNA and DNA quasispecies in most nt positions [218/357 (61.06%)]. In 102 of the remaining 139 (73.38%), HBV-RNA showed slightly higher variability. Sliding window analysis identified 4 hyper-conserved sequence fragments in each quasispecies, 3 of them coincided between the 2 quasispecies: nts 1258-1286, 1545-1573 and 1575-1604. The 2 hyper-variable sequence fragments also coincided: nts 1311-1344 and 1461-1485. Sequences between nts 1519-1543 and 1559-1587 were only hyper-conserved in HBV-DNA and RNA, respectively. CONCLUSION: Our methodology allowed analyzing HBV-RNA quasispecies complexity and conservation without interference from HBV-DNA. Thanks to this, we have been able to compare both quasispecies in the present study.

Study of Quasispecies Complexity and Liver Damage Progression after Liver Transplantation in Hepatitis C Virus Infected Patients.

Cirrhosis derived from chronic hepatitis C virus (HCV) infection is still a common indication for liver transplantation (LT). Reinfection of the engrafted liver is universal in patients with detectable viral RNA at the time of transplant and causes fast progression of cirrhosis (within 5 years) in around one-third of these patients. To prevent damage to the liver graft, effective direct-acting antiviral (DAA) therapy is required as soon as possible. However, because of post-LT clinical instability, it is difficult to determine the optimal time to start DAAs with a low risk of complications. Evaluate changes in quasispecies complexity following LT and seek a predictive index of fast liver damage progression to determine the timing of DAA initiation. HCV genomes isolated from pre-LT and 15-day post-LT serum samples of ten patients, who underwent orthotopic LT, were quantified and sequenced using a next-generation sequencing platform. Sequence alignments, phylogenetic trees, quasispecies complexity measures, biostatistics analyses, adjusted R2 values, and analysis of variance (ANOVA) were carried out. Three different patterns of reinfection were observed (viral bottlenecking, conserved pre-LT population, and mixed populations), suggesting that bottlenecking or homogenization of the viral population is not a generalized effect after liver graft reinfection. None of the quasispecies complexity measures predicted the future degree of liver damage. Higher and more uniform viral load (VL) values were observed in all pre-LT samples, but values were more dispersed in post-LT samples. However, VL increased significantly from the pre-LT to 15-day post-LT samples in patients with advanced fibrosis at 1-year post-LT, suggesting that a VL increase on day 15 may be a predictor of fast liver fibrosis progression. HCV kinetics after LT differ between patients and are not fibrosis-dependent. Higher VL at day 15 post-LT versus pre-LT samples may predict fast liver fibrosis progression.

Monitoring Emergence of the SARS-CoV-2 B.1.1.7 Variant through the Spanish National SARS-CoV-2 Wastewater Surveillance System (VATar COVID-19).

Since its first identification in the United Kingdom in late 2020, the highly transmissible B.1.1.7 variant of SARS-CoV-2 has become dominant in several countries raising great concern. We developed a duplex real-time RT-qPCR assay to detect, discriminate, and quantitate SARS-CoV-2 variants containing one of its mutation signatures, the ΔHV69/70 deletion, and used it to trace the community circulation of the B.1.1.7 variant in Spain through the Spanish National SARS-CoV-2 Wastewater Surveillance System (VATar COVID-19). The B.1.1.7 variant was detected earlier than clinical epidemiological reporting by the local authorities, first in the southern city of Málaga (Andalucía) in week 20_52 (year_week), and multiple introductions during Christmas holidays were inferred in different parts of the country. Wastewater-based B.1.1.7 tracking showed a good correlation with clinical data and provided information at the local level. Data from wastewater treatment plants, which reached B.1.1.7 prevalences higher than 90% for ≥2 consecutive weeks showed that 8.1 ± 2.0 weeks were required for B.1.1.7 to become dominant. The study highlights the applicability of RT-qPCR-based strategies to track specific mutations of variants of concern as soon as they are identified by clinical sequencing and their integration into existing wastewater surveillance programs, as a cost-effective approach to complement clinical testing during the COVID-19 pandemic.

Host-dependent editing of SARS-CoV-2 in COVID-19 patients.

A common trait among RNA viruses is their high capability to acquire genetic variability due to viral and host mechanisms. Next-generation sequencing (NGS) analysis enables the deep study of the viral quasispecies in samples from infected individuals. In this study, the viral quasispecies complexity and single nucleotide polymorphisms of the SARS-CoV-2 spike gene of coronavirus disease 2019 (COVID-19) patients with mild or severe disease were investigated using next-generation sequencing (Illumina platform). SARS-CoV-2 spike variability was higher in patients with long-lasting infection. Most substitutions found were present at frequencies lower than 1%, and had an A → G or T → C pattern, consistent with variants caused by adenosine deaminase acting on RNA-1 (ADAR1). ADAR1 affected a small fraction of replicating genomes, but produced multiple, mainly non-synonymous mutations. ADAR1 editing during replication rather than the RNA-dependent RNA polymerase (nsp12) was the predominant mechanism generating SARS-CoV-2 genetic variability. However, the mutations produced are not fixed in the infected human population, suggesting that ADAR1 may have an antiviral role, whereas nsp12-induced mutations occurring in patients with high viremia and persistent infection are the main source of new SARS-CoV-2 variants.