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Worldwide outbreaks of enterovirus D68 (EV-D68) in 2014 and 2016 have caused serious respiratory and neurological disease. We collected samples from several European countries during the 2018 outbreak and determined 53 near full-length genome ('whole genome') sequences. These sequences were combined with 718 whole genome and 1,987 VP1-gene publicly available sequences. In 2018, circulating strains clustered into multiple subgroups in the B3 and A2 subclades, with different phylogenetic origins. Clusters in subclade B3 emerged from strains circulating primarily in the US and Europe in 2016, though some had deeper roots linking to Asian strains, while clusters in A2 traced back to strains detected in East Asia in 2015-2016. In 2018, all sequences from the USA formed a distinct subgroup, containing only three non-US samples. Alongside the varied origins of seasonal strains, we found that diversification of these variants begins up to 18 months prior to the first diagnostic detection during a EV-D68 season. EV-D68 displays strong signs of continuous antigenic evolution and all 2018 A2 strains had novel patterns in the putative neutralizing epitopes in the BC- and DE-loops. The pattern in the BC-loop of the USA B3 subgroup had not been detected on that continent before. Patients with EV-D68 in subclade A2 were significantly older than patients with a B3 subclade virus. In contrast to other subclades, the age distribution of A2 is distinctly bimodal and was found primarily among children and in the elderly. We hypothesize that EV-D68's rapid evolution of surface proteins, extensive diversity, and high rate of geographic mixing could be explained by substantial reinfection of adults. Better understanding of evolution and immunity across diverse viral pathogens, including EV-D68 and SARS-CoV-2, is critical to pandemic preparedness in the future.

ABSTRACTThe emergence of the Omicron SARS-CoV-2 variant of concern has changed the COVID-19 scenario as this variant is characterized by high transmissibility and immune evasion ability. To evaluate the impact of this variant on the Canary Islands (Spain) population, we determined the reinfection rates and disease severity associated with the Omicron sublineages and the previously circulating variants of concern. We performed a retrospective observational study on 21,745 SARS-CoV-2 viral genomes collected from December 2020 to July 2022 in the Canary Islands (Spain). We compared the reinfection rates between lineages using pairwise proportion and Fisher’s exact tests. To assess disease severity, we studied the association of Alpha, Delta, BA.1, BA.2, BA.5, and other risk factors on 28-day hospital mortality using logistic regression and Cox proportional hazard models. We observed 127 bona fide reinfection cases throughout the study period. We found that BA.5 had the highest reinfection rate compared to other lineages (vs. Delta p = 2.89 × 10−25; vs. BA.1 p = 5.17 × 10−11; vs. BA.2 p = 0.002). Among the 1,094 hospitalized patients, multivariate logistic regression showed that Alpha (Odds Ratio [OR] = 0.45, 95% Confidence Interval [CI] = 0.23-0.87, p = 0.02), BA.2 (OR = 0.38, 95% CI = 0.22-0.63, p = 1.91 × 10−4), and BA.5 (OR = 0.30, 95% CI = 0.16-0.55, p = 1.05 × 10−4) had lower 28-day hospital mortality compared to Delta. These results were confirmed by using Cox proportional hazard models. Omicron lineages, and in particular BA.5, were associated with higher reinfection rates and lower disease severity (28-day hospital mortality) than previously circulating variants of concern.

On July 23, 2022, monkeypox disease (mpox) was declared a Public Emergency of International Concern (PHEIC) by the World Health Organization (WHO) due to a multicountry outbreak. In Europe, several cases of mpox virus (MPXV) infection related to this outbreak were detected in the Canary Islands (Spain). Here we describe the combination of viral DNA sequencing and bioinformatic approaches, including methods for de novo genome assembly and short- and long-read technologies, used to reconstruct the first MPXV genome isolated in the Canary Islands on the 31st of May 2022 from a male adult patient with mild symptoms. The same sequencing and bioinformatic approaches were then validated with three other positive cases of MPXV infection from the same mpox outbreak. We obtained the best results using a reference-based approach with short reads, evidencing 46–79 nucleotide variants against viral sequences from the 2018–2019 mpox outbreak and placing the viral sequences in the new B.1 sublineage of clade IIb of the MPXV classification. This study of MPXV demonstrates the potential of metagenomics sequencing for rapid and precise pathogen identification.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the associated coronavirus disease 2019 (COVID-19), which severely affect the respiratory system and several organs and tissues, and may lead to death, have shown how science can respond when challenged by a global emergency, offering as a response a myriad of rapid technological developments. Development of vaccines at lightning speed is one of them. SARS-CoV-2 outbreaks have stressed healthcare systems, questioning patients care by using standard non-adapted therapies and diagnostic tools. In this scenario, nanotechnology has offered new tools, techniques and opportunities for prevention, for rapid, accurate and sensitive diagnosis and treatment of COVID-19. In this review, we focus on the nanotechnological applications and nano-based materials (i.e., personal protective equipment) to combat SARS-CoV-2 transmission, infection, organ damage and for the development of new tools for virosurveillance, diagnose and immune protection by mRNA and other nano-based vaccines. All the nano-based developed tools have allowed a historical, unprecedented, real time epidemiological surveillance and diagnosis of SARS-CoV-2 infection, at community and international levels. The nano-based technology has help to predict and detect how this Sarbecovirus is mutating and the severity of the associated COVID-19 disease, thereby assisting the administration and public health services to make decisions and measures for preparedness against the emerging variants of SARS-CoV-2 and severe or lethal COVID-19.

Several variants of concern (VOCs) explain most of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic waves in Europe. We aimed to dissect the spread of the SARS-CoV-2 VOCs in the Canary Islands (Spain) between December 2020 and September 2021 at a micro-geographical level. We sequenced the viral genome of 8,224 respiratory samples collected in the archipelago. We observed that Alpha (B.1.1.7) and Delta (B.1.617.2 and sublineages) were ubiquitously present in the islands, while Beta (B.1.351) and Gamma (P.1/P.1.1) had a heterogeneous distribution and were responsible for fewer and more controlled outbreaks. This work represents the largest effort for viral genomic surveillance in the Canary Islands so far, helping the public health bodies in decision-making throughout the pandemic.

Millions of SARS-CoV-2 whole genome sequences have been generated to date. However, good quality data and adequate surveillance systems are required to contribute to meaningful surveillance in public health. In this context, the network of Spanish laboratories for coronavirus (RELECOV) was created with the main goal of promoting actions to speed up the detection, analyses, and evaluation of SARS-CoV-2 at a national level, partially structured and financed by an ECDC-HERA-Incubator action (ECDC/GRANT/2021/024). A SARS-CoV-2 sequencing quality control assessment (QCA) was developed to evaluate the network’s technical capacity. QCA full panel results showed a lower hit rate for lineage assignment compared to that obtained for variants. Genomic data comprising 48,578 viral genomes were studied and evaluated to monitor SARS-CoV-2. The developed network actions showed a 36% increase in sharing viral sequences. In addition, analysis of lineage/sublineage-defining mutations to track the virus showed characteristic mutation profiles for the Delta and Omicron variants. Further, phylogenetic analyses strongly correlated with different variant clusters, obtaining a robust reference tree. The RELECOV network has made it possible to improve and enhance the genomic surveillance of SARS-CoV-2 in Spain. It has provided and evaluated genomic tools for viral genome monitoring and characterization that make it possible to increase knowledge efficiently and quickly, promoting the genomic surveillance of SARS-CoV-2 in Spain.

Objectives: Limited testing capacity has characterized the ongoing coronavirus disease 2019 (COVID-19) pandemic in Spain, hampering timely control of outbreaks and opportunities to reduce the escalation of community transmission. This study investigated the potential to use sample pooling, followed by one-step retrotranscription and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) to increase testing capacity for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Methods: Various pool sizes (five, 10 and 15 samples) were evaluated prior to RNA extraction followed by standard RT-qPCR for the diagnosis of COVID-19. The pool size achieving reproducible results with individual sample testing was subsequently used to assess nasopharyngeal samples in a tertiary hospital in August 2020. Results: A pool size of five samples had higher sensitivity compared with pool sizes of 10 and 15 samples, showing a mean cycle threshold (Ct) shift of 3.5 [standard deviation (SD) 2.2] between the pooled test and positive samples in the pool. Next, a pool size of five was used to test a total of 895 pools (4475 prospective samples) using two different RT-qPCR kits. The Real Accurate Quadruplex corona-plus PCR Kit (PathoFinder) reported the lowest mean Ct shift [2.2 (SD 2.4)] between the pool and individual samples. This strategy enables detection of individual positive samples in positive pools with Ct of 16.7–39.4. Conclusions: Grouping samples into pools of five for RT-qPCR resulted in an increase in SARS-CoV-2 testing capacity with minimal loss of sensitivity compared with testing each sample individually.

Objectives: The ongoing COVID-19 pandemic continues to impose demands on diagnostic screening. In anticipation that the recurrence of outbreaks and the measures for lifting the lockdown worldwide may cause supply chain issues over the coming months, this study assessed the sensitivity of a number of one-step retrotranscription and quantitative polymerase chain reaction (RT-qPCR) solutions to detect SARS-CoV-2. Methods: Six different RT-qPCR alternatives were evaluated for SARS-CoV-2/COVID-19 diagnosis based on standard RNA extractions. The one with best sensitivity was also assessed with direct nasopharyngeal swab viral transmission medium (VTM) heating; thus overcoming the RNA extraction step. Results: A wide variability in the sensitivity of RT-qPCR solutions was found that was associated with a range of false negatives from 2% (0.3–7.9%) to 39.8% (30.2–50.2%). Direct preheating of VTM combined with the best solution provided a sensitivity of 72.5% (62.5–81.0%), in the range of some of the solutions based on standard RNA extractions. Conclusions: Sensitivity limitations of currently used RT-qPCR solutions were found. These results will help to calibrate the impact of false negative diagnoses of COVID-19, and to detect and control new SARS-CoV-2 outbreaks and community transmissions.

Objectives: The gold-standard COVID-19 diagnosis relies on detecting SARS-CoV-2 using RNA purification and one-step retrotranscription and quantitative PCR (RT-qPCR). Based on the urgent need for high-throughput screening, we tested the performance of three alternative, simple and affordable protocols to rapidly detect SARS-CoV-2, bypassing the long and tedious RNA extraction step and reducing the time to viral detection. Methods: We evaluated three methods based on direct nasopharyngeal swab viral transmission medium (VTM) heating before the RT-qPCR: a) direct without additives; b) in a formamide-EDTA (FAE) buffer, c) in a RNAsnapTM buffer. Results: Although with a delay in cycle threshold compared to the gold-standard, we found consistent results in nasopharyngeal swab samples that were subject to a direct 70°C incubation for 10 min. Conclusions: Our findings provide valuable options to overcome any supply chain issue and help to increase the throughput of diagnostic tests, thereby complementing standard diagnosis.

DNA methylation clocks are used as molecular estimators of epigenetic age, but with little evidence in mothers and none in neglectful mothering. We investigated differences in epigenetic age acceleration (EAA) and the role of empathy using the PhenoAge clock. We collected saliva samples from mothers with extreme disregard for their child’s needs (50 in the neglect group, NG) and mothers with non-neglectful caregiving (87 in the control group, CG). Mothers completed an empathy scale, along with questionnaires of their own childhood maltreatment, adverse life events and psychiatric disorders. Sociodemographic variables potentially affecting EAA were also measured. The ANCOVA solution showed a significant increase in EAA in the NG compared to the CG, after adjustment for maternal age, number of pregnancies, financial assistance, adverse events, childhood maltreatment and psychiatric disorder. The group interaction effects showed a reduction in EAA for greater empathic concern and for a higher education level both as positive factors, and an increment in EAA for mothers living in a two-parent family as a risk factor, all in the NG. Our findings open the search for protective factors of EAA associated with caregiver behavior to reduce health vulnerabilities and poor social functioning, especially for mothers at risk of maladaptive caregiving.