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.

Genomic evolution of human respiratory syncytial virus during a decade (2013-2023): bridging the path to monoclonal antibody surveillance.

OBJECTIVES: This study investigated the prevalence, genetic diversity, and evolution of human respiratory syncytial virus (HRSV) in Barcelona from 2013 to 2023. METHODS: Respiratory specimens from patients with RTI suspicion at Hospital Universitari Vall d'Hebron were collected from October 2013 to May 2023 for laboratory-confirmation of respiratory viruses. Next-generation sequencing was performed in randomly-selected samples with Illumina technology. Phylogenetic analyses of whole genome sequences were performed with BEAST v1.10.4. Signals of selection and evolutionary pressures were inferred by population dynamics and evolutionary analyses. Mutations in major surface proteins were genetic and structurally characterised, emphasizing those within antigenic epitopes. RESULTS: Analyzing 139,625 samples, 5.3% were HRSV-positive (3008 HRSV-A, 3882 HRSV-B, 56 HRSV-A and -B, and 495 unsubtyped HRSV), with a higher prevalence observed in the paediatric population. Pandemic-related shifts in seasonal patterns returned to normal in 2022-2023. A total of 198 whole-genome sequences were obtained for HRSV-A (6.6% of the HRSV-A positive samples) belonging to GA2.3.5 lineage. For HRSV-B, 167 samples were sequenced (4.3% of the HRSV-B positive samples), belonging to GB5.0.2, GB5.0.4a and GB5.0.5a. HRSV-B exhibited a higher evolution rate. Post-SARS-CoV-2 pandemic, both subtypes showed increased evolutionary rates and decreased effective population size initially, followed by a sharp increase. Analyses indicated negative selective pressure on HRSV. Mutations in antigenic epitopes, including S276N and M274I in palivizumab-targeted site II, and I206M, Q209R, and S211N in nirsevimab-targeted site Ø, were identified. DISCUSSION: Particularly in the context of the large-scale use in 2023-2024 season of nirsevimab, continuous epidemiological and genomic surveillance is crucial.

Suspected case of monkeypox reinfection versus reactivation in a immunocompetent patient, Barcelona, 2022.

Vaccines against smallpox are known to have cross-protective activity against monkeypox, and smallpox and monkeypox infections are believed to generate permanent immunity. Nevertheless, there are scarce data about the possibility of reinfection or reactivation. Recently, a case of apparent monkeypox reinfection has been reported. We present a suspected case of second episode of monkeypox in a healthy and previously vaccinated man, with a confirmed primary monkeypox infection occurring three months before the second confirmed presentation.

The emergence, impact, and evolution of human metapneumovirus variants from 2014 to 2021 in Spain.

BACKGROUND: Human metapneumovirus (HMPV) is an important aetiologic agent of respiratory tract infection (RTI). This study aimed to describe the prevalence, genetic diversity, and evolutionary dynamics of HMPV. METHODS: Laboratory-confirmed HMPV were characterised based on partial-coding G gene sequences with MEGA.v6.0. WGS was performed with Illumina, and evolutionary analyses with Datamonkey and Nextstrain. RESULTS: HMPV prevalence was 2.5%, peaking in February-April and with an alternation in the predominance of HMPV-A and -B until the emergence of SARS-CoV-2, not circulating until summer and autumn-winter 2021, with a higher prevalence and with the almost only circulation of A2c111dup. G and SH proteins were the most variable, and 70% of F protein was under negative selection. Mutation rate of HMPV genome was 6.95 × 10-4 substitutions/site/year. CONCLUSION: HMPV showed a significant morbidity until the emergence of SARS-CoV-2 pandemic in 2020, not circulating again until summer and autumn 2021, with a higher prevalence and with almost the only circulation of A2c111dup, probably due to a more efficient immune evasion mechanism. The F protein showed a very conserved nature, supporting the need for steric shielding. The tMRCA showed a recent emergence of the A2c variants carrying duplications, supporting the importance of virological surveillance.

Detection of reassortant influenza B strains from 2004 to 2015 seasons in Barcelona (Catalonia, Spain) by whole genome sequencing.

BACKGROUND: Influenza B viruses (FLUBV) have segmented genomes which enables the virus to evolve by segment reassortment. Since the divergence of both FLUBV lineages, B/Victoria/2/87 (FLUBV/VIC) and B/Yamagata/16/88 (FLUBV/YAM), PB2, PB1 and HA have kept the same ancestor, while some reassortment events in the other segments have been reported worldwide. The aim of the present study was to find out reassortment episodes in FLUBV strains detected in cases attended at Hospital Universitari Vall d'Hebron and Hospital de la Santa Creu i Sant Pau (Barcelona, Spain) from 2004 to 2015 seasons. METHODS: From October 2004 to May 2015, respiratory specimens were received from patients with respiratory tract infection suspicion. Influenza detection was carried out by either cell culture isolation, immunofluorescence or PCR-based assays. A RT-PCR was performed to distinguish both lineages by agarose gel electrophoresis. Whole genome amplification was performed using the universal primer set by Zhou et al. in 2012, and subsequently sequenced using Roche 454 GS Junior platform. Bioinformatic analysis was performed to characterise the sequences with B/Malaysia/2506/2007 and B/Florida/4/2006 corresponding sequences as reference of (B/VIC) and (B/YAM), respectively. RESULTS: A total of 118 FLUBV (75 FLUBV/VIC and 43 FLUBV/YAM), from 2004 to 2006, 2008-2011 and 2012-2015 seasons, were studied. The whole genome of 58 FLUBV/VIC and 42 FLUBV/YAM viruses was successfully amplified. Based on HA sequences, most FLUBV/VIC viruses (37; 64%) belonged to clade 1A (B/Brisbane/60/2008) except to 11 (19%), which fell within clade 1B (B/HongKong/514/2009) and 10 (17%) to B/Malaysia/2506/2004. Nine (20%) FLUBV/YAM viruses belonged to clade 2 (B/Massachusetts/02/2012), 18 (42%) to clade 3 (B/Phuket/3073/2013) and 15 (38%) fell within Florida/4/2006. Numerous intra-lineage reassortments in PB2, PB1, NA and NS were found in 2 2010-2011 viruses. An important inter-lineage reassortment event from 2008 to 2009 (11), 2010-2011 (26) and 2012-2013 (3) FLUBV/VIC (clade 1) strains to FLUBV/YAM (clade 3) was found, in addition to 1 reassortant NS in 2010-2011 B/VIC virus. CONCLUSIONS: Intra- and inter-lineage reassortment episodes were revealed by WGS. While PB2-PB1-HA remained in complex, NP and NS reassortant viruses were found in both lineages. Despite reassorment events are not often, the characterisation only by HA and NA sequences might be underestimating their detection.

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.

The burden of non-SARS-CoV2 viral lower respiratory tract infections in hospitalized children in Barcelona (Spain): A long-term, clinical, epidemiologic and economic study.

BACKGROUND: Viral lower respiratory tract infections (LRTI) are the leading cause of hospitalization in children. In Catalonia (Spain), information is scarce about the burden of viral LRTIs in paediatric hospitalizations. The aim of this study is to describe epidemiological, clinical, virological and economic features of paediatric hospitalizations due to viral LRTI. METHODS: From October 2012 to December 2020, children aged <16 years admitted to a tertiary paediatric hospital in Catalonia (Spain) with confirmed viral LRTI were included in the study. Virus seasonality, prevalence, age and sex distribution, clinical characteristics, hospital costs and bed occupancy rates were determined. RESULTS: A total of 3,325 children were included (57.17% male, 9.44% with comorbidities) accounting for 4056 hospitalizations (32.47% ≤ 12 months): 53.87% with wheezing/asthma, 37.85% with bronchiolitis and 8.28% with pneumonia. The most common virus was respiratory syncytial virus (RSV) (52.59%). Influenza A was associated with pneumonia (odds ratio [OR] 7.75) and caused longer hospitalizations (7 ± 31.58 days), while RSV was associated with bronchiolitis (OR 6.62) and was the most frequent reason for admission to the paediatric intensive care unit (PICU) (11.23%) and for respiratory support (78.76%). Male sex, age ≤12 months, chronic conditions and bronchiolitis significantly increased the odds of PICU admission. From October to May, viral LRTIs accounted for 12.36% of overall hospital bed days. The total hospitalization cost during the study period was €16,603,415. CONCLUSIONS: Viral LRTIs are an important cause of morbidity, hospitalization and PICU admission in children. The clinical burden is associated with significant bed occupancy and health-care costs, especially during seasonal periods.

Molecular characterization and clinical impact of human bocavirus at a tertiary hospital in Barcelona (Catalonia, Spain) during the 2014-2017 seasons.

PURPOSE: The aim was to describe the prevalence, molecular epidemiology and clinical manifestations of human bocavirus (HBoV) in patients attended at a tertiary hospital in Barcelona, Spain. METHODS: From October 2014 to May 2017, respiratory specimens from paediatric patients were collected for respiratory viruses' laboratory-confirmation. Phylogenetic analyses from partial VP1 sequences were performed from all HBoV laboratory-confirmed specimens. Clinical features were retrospectively studied. RESULTS: 178/10271 cases were HBoV laboratory-confirmed. The median age was 1.53 (IQR 1.0-2.3). Co-detection was highly reported (136; 76%). All viruses belonged into HBoV1 genotype but one into HBoV2. Non-reported mutations were observed and two sites were suggestive to be under negative selection. 61% (109/178) cases had lower RTI (LRTI), of whom 84 had co-detections (77%) and 76 had comorbidities (70%). LRTI was the cause of hospitalization in 85 out of 109 cases (78%), and no differences were found regarding severity factors during hospitalization between co- and single-detections, except for median length of respiratory support, which was longer in cases with co-detections. CONCLUSIONS: Close monitoring of predominant HBoV1 showed a high similarity between viruses. The presence of comorbidities might explain the high prevalence of LRTI. Symptomatology in HBoV single-detected cases suggest that HBoV is a true pathogen.

Emergence of Delta and Omicron variants carrying resistance-associated mutations in immunocompromised patients undergoing sotrovimab treatment with long-term viral excretion.

OBJECTIVES: To monitor the early emergence of genetic mutations related to reduced susceptibility to monoclonal anti-body (mAb)-based treatment in immunocompromised patients with long-term viral excretion using whole-genome sequencing at a tertiary university hospital in Barcelona, Spain. METHODS: Serial severe acute respiratory syndrome coronavirus 2-positive samples (mid-December 2021-mid-March 2022) from eight immunosuppressed, fully vaccinated patients (for solid-organ transplantation or haematologic malignancies) with long-term viral excretion despite undergoing mAb therapy (sotrovimab) for coronavirus disease 2019 were selected. Whole-genome sequencing was performed following the ARTIC, version 4.1, protocol on the MiSeq platform. Mutations in the coding sequence of the spike protein with a frequency of ≥5% were studied. RESULTS: A total of 37 samples from the studied cases were analysed. All the cases, except one, were confirmed to have the Omicron variant BA.1; one had Delta (AY.100). Thirty-four different mutations were detected within the receptor-binding domain of the spike protein in 62.5% of patients, eight of which were not lineage related and located in the sotrovimab target epitope (P337L, E340D, E340R, E340K, E340V, E340Q, R346T and K356T). Except for P337L, all changes showed a significant increase in frequency or fixation after the administration of sotrovimab. Some of them have been associated with either reduced susceptibility to mAb therapy, such as those at position 340, or the acquisition of a new glycosylation site (346 and 356 positions). CONCLUSIONS: This study highlights the importance of monitoring for early in vivo selection of mutations associated with reduced susceptibility to mAb therapy, especially in immunocompromised patients receiving anti-viral drugs, whose immune response is not able to control viral replication, resulting in long-term viral shedding, and those receiving selective evolution pressure. Virologic surveillance of genetically resistant viruses to available anti-viral therapies is considered a priority for both patients and the community.

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.

Clinical presentation and virological assessment of confirmed human monkeypox virus cases in Spain: a prospective observational cohort study.

BACKGROUND: In May, 2022, several European countries reported autochthonous cases of monkeypox, which rapidly spread globally. Early reports suggest atypical presentations. We aimed to investigate clinical and virological characteristics of cases of human monkeypox in Spain. METHODS: This multicentre, prospective, observational cohort study was done in three sexual health clinics in Madrid and Barcelona, Spain. We enrolled all consecutive patients with laboratory-confirmed monkeypox from May 11 to June 29, 2022. Participants were offered lesion, anal, and oropharynx swabs for PCR testing. Participant data were collected by means of interviews conducted by dermatologists or specialists in sexually transmitted infections and were recorded using a standard case report form. Outcomes assessed in all participants with a confirmed diagnosis were demographics, smallpox vaccination, HIV status, exposure to someone with monkeypox, travel, mass gathering attendance, risk factors for sexually transmitted infections, sexual behaviour, signs and symptoms on first presentation, virological results at multiple body sites, co-infection with other sexually transmitted pathogens, and clinical outcomes 14 days after the initial presentation. Clinical outcomes were followed up until July 13, 2022. FINDINGS: 181 patients had a confirmed monkeypox diagnosis and were enrolled in the study. 166 (92%) identified as gay men, bisexual men, or other men who have sex with men (MSM) and 15 (8%) identified as heterosexual men or heterosexual women. Median age was 37·0 years (IQR 31·0-42·0). 32 (18%) patients reported previous smallpox vaccination, 72 (40%) were HIV-positive, eight (11%) had a CD4 cell count less than 500 cells per µL, and 31 (17%) were diagnosed with a concurrent sexually transmitted infection. Median incubation was 7·0 days (IQR 5·0-10·0). All participants presented with skin lesions; 141 (78%) participants had lesions in the anogenital region, and 78 (43%) in the oral and perioral region. 70 (39%) participants had complications requiring treatment: 45 (25%) had a proctitis, 19 (10%) had tonsillitis, 15 (8%) had penile oedema, six (3%) an abscess, and eight (4%) had an exanthem. Three (2%) patients required hospital admission. 178 (99%) of 180 swabs from skin lesions collected tested positive, as did 82 (70%) of 117 throat swabs. Viral load was higher in lesion swabs than in pharyngeal specimens (mean cycle threshold value 23 [SD 4] vs 32 [6], absolute difference 9 [95% CI 8-10]; p<0·0001). 108 (65%) of 166 MSM reported anal-receptive sex. MSM who engaged in anal-receptive sex presented with proctitis (41 [38%] of 108 vs four [7%] of 58, absolute difference 31% [95% CI 19-44]; p<0·0001) and systemic symptoms before the rash (67 [62%] vs 16 [28%], absolute difference 34% [28-62]; p<0·0001) more frequently than MSM who did not engage in anal-receptive sex. 18 (95%) of 19 participants with tonsillitis reported practising oral-receptive sex. The median time from onset of lesions to formation of a dry crust was 10 days (IQR 7-13). INTERPRETATION: In our cohort, monkeypox caused genital, perianal, and oral lesions and complications including proctitis and tonsillitis. Because of the variability of presentations, clinicians should have a low threshold for suspicion of monkeypox. Lesion swabs showed the highest viral loads, which, combined with the history of sexual exposure and the distribution of lesions, suggests close contact is probably the dominant transmission route in the current outbreak. FUNDING: None.

Characteristics of 24 SARS-CoV-2-Sequenced Reinfection Cases in a Tertiary Hospital in Spain.

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.

Enterovirus D68 in Hospitalized Children, Barcelona, Spain, 2014-2021.

To determine molecular epidemiology and clinical features of enterovirus D68 (EV-D68) infections, we reviewed EV-D68-associated respiratory cases at a hospital in Barcelona, Spain, during 2014-2021. Respiratory samples were collected from hospitalized patients or outpatients with symptoms of acute respiratory tract infection or suggestive of enterovirus infection. Enterovirus detection was performed by real-time multiplex reverse transcription PCR and characterization by phylogenetic analysis of the partial viral protein 1 coding region sequences. From 184 patients with EV-D68 infection, circulating subclades were B3 (80%), D1 (17%), B2 (1%), and A (<1%); clade proportions shifted over time. EV-D68 was detected mostly in children (86%) and biennially (2016, 2018, 2021). In patients <16 years of age, the most common sign/symptom was lower respiratory tract infection, for which 11.8% required pediatric intensive care unit admission and 2.3% required invasive mechanical ventilation; neurologic complications developed in 1. The potential neurotropism indicates that enterovirus surveillance should be mandatory.

Next-Generation Sequencing for Confronting Virus Pandemics.

Virus pandemics have happened, are happening and will happen again. In recent decades, the rate of zoonotic viral spillover into humans has accelerated, mirroring the expansion of our global footprint and travel network, including the expansion of viral vectors and the destruction of natural spaces, bringing humans closer to wild animals. Once viral cross-species transmission to humans occurs, transmission cannot be stopped by cement walls but by developing barriers based on knowledge that can prevent or reduce the effects of any pandemic. Controlling a local transmission affecting few individuals is more efficient that confronting a community outbreak in which infections cannot be traced. Genetic detection, identification, and characterization of infectious agents using next-generation sequencing (NGS) has been proven to be a powerful tool allowing for the development of fast PCR-based molecular assays, the rapid development of vaccines based on mRNA and DNA, the identification of outbreaks, transmission dynamics and spill-over events, the detection of new variants and treatment of vaccine resistance mutations, the development of direct-acting antiviral drugs, the discovery of relevant minority variants to improve knowledge of the viral life cycle, strengths and weaknesses, the potential for becoming dominant to take appropriate preventive measures, and the discovery of new routes of viral transmission.

Clinical evaluation of the Procleix SARS-CoV-2 assay, a sensitive, high-throughput test that runs on an automated system.

Testing is crucial in controlling COVID-19. The Procleix® SARS-CoV-2 assay, a transcription-mediated amplification nucleic acid test that runs on an automated system, was evaluated using inactivated virus and clinical samples. The sensitivity of the assay was assessed using heat-inactivated SARS-CoV-2 and compared to 3 other tests. Clinical validation utilized 2 sets of samples: (1) Nasal, nasopharyngeal and oropharyngeal samples (n = 963) from asymptomatic individuals, and (2) nasopharyngeal samples from symptomatic patients: 100 positive and 100 negative by RT-PCR. The Procleix assay had greater sensitivity (3-fold to 100-fold) than the comparators and had high specificity (100%) in asymptomatic subjects. In symptomatic patients, the Procleix assay detected 100% of PCR-positives and found 24 positives in the initial PCR-negatives. Eighteen of these were confirmed positive and 6 were inconclusive. These studies showed that the Procleix SARS-CoV-2 assay was a sensitive and specific tool for detecting COVID-19.

A year living with SARS-CoV-2: an epidemiological overview of viral lineage circulation by whole-genome sequencing in Barcelona city (Catalonia, Spain).

Herein, we describe the genetic diversity of circulating SARS-CoV-2 viruses by whole-genome sequencing (WGS) in Barcelona city (Catalonia, Spain) throughout the first four pandemic waves. From weeks 11/2020-24/2021, SARS-CoV-2-positive respiratory samples were randomly selected per clinical setting (80% from primary care or 20% from the hospital), age group, and week. WGS was performed following the ARTICv3 protocol on MiSeq or NextSeq2000 Illumina platforms. Nearly complete consensus sequences were used for genetic characterization based on GISAID and PANGOLIN nomenclatures. From 2475 samples, 2166 (87%) were fully sequenced (78% from primary care and 22% from hospital settings). Multiple genetic lineages were co-circulating, but four were predominant at different periods. While B.1.5 (50.68%) and B.1.1 (32.88%) were the major lineages during the first pandemic wave, B.1.177 (66.85%) and B.1.1.7 (83.80%) were predominant during the second, third, and fourth waves, respectively. Almost all (96.4%) were carrying D614G mutation in the S protein, with additional mutations that define lineages or variants. But some mutations of concern, such as E484K from B.1.351 and P.1 lineages are currently under monitoring, together with those observed in the receptor-binding domain or N-terminal domain, such as L452R and T478K from B.1.617.2 lineage. The fact that a predominant lineage was observed in each pandemic wave suggests advantageous properties over other contemporary co-circulating variants. This genetic variability should be monitored, especially when a massive vaccination campaign is ongoing because the potential selection and emergence of novel antigenic SARS-CoV-2 strains related to immunological escapement events.

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.

Severe SARS-CoV-2 placenta infection can impact neonatal outcome in the absence of vertical transmission.

The effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the pathophysiology of the placenta and its impact on pregnancy outcome has not yet been fully elucidated. Here, we present a comprehensive clinical, morphological, and molecular analysis of placental tissues from pregnant women with and without SARS-CoV-2 infection. SARS-CoV-2 could be detected in half of placental tissues from SARS-CoV-2-positive women. The presence of the virus was not associated with any distinctive pathological, maternal, or neonatal outcome features. SARS-CoV-2 tissue load was low in all but one patient who exhibited severe placental damage leading to neonatal neurological manifestations. The placental transcriptional response induced by high viral load of SARS-CoV-2 showed an immunopathology phenotype similar to autopsy lung tissues from patients with severe coronavirus disease 2019. This finding contrasted with the lack of inflammatory response in placental tissues from SARS-CoV-2-positive women with low viral tissue load and from SARS-CoV-2-negative women. Importantly, no evidence of vertical transmission of SARS-CoV-2 was found in any newborns, suggesting that the placenta may be an effective maternal-neonatal barrier against the virus even in the presence of severe infection. Our observations suggest that severe placental damage induced by the virus may be detrimental for the neonate independently of vertical transmission.