José María González-Alba

The ability of a virus to adapt is key to its survival, and this is achieved through mutation, which allows the virus to change and adapt to new environments. To capture the full extent of SARS-CoV-2 diversity in Asturias, samples obtained from nasopharyngeal swabs were characterised using whole-genome sequencing. Between 2020 and July 2024, a total of 4001 sequences were analysed and 5302 mutations were identified. An increase in the positivity rate was observed between 2022 and 2024 in children under 1 year of age. During this period, 55 new circulating variants belonging to 41 pangolin lineages were detected: 24 originated throughout the world and 31 in Asturias (10 detected only in the region, 8 in the rest of Spain, and 13 around the world). A total of 31 new non-synonymous mutations were fixed in the viral population 250 ± 46 (93–620) days after their appearance. During seasonal SARS-CoV-2 circulation, surveillance systems developed during the pandemic continue to detect new indigenous and imported variants without indicating an increase in severity.

Background: The mutation of a new virus is key to its ability to adapt, as it allows the virus to change and survive in new environments. We try to capture the full extent of SARS-CoV-2 diversity in Asturias. Methods: Samples from nasopharyngeal swabs belonging to SARS-CoV-2 infected patients were characterized by the whole genome sequencing. Results: A total of 4,001 sequences were analysed between 2020 and July 2024. During this time, 5302 mutations were found (157 occurred in more than 5% of the viral population). Between 2022 and 2024, 88 new non-synonymous mutations occurred in over 5% of the viral population, of which 31 are currently maintained and were fixed 250±46 (93-620) days after their appearance. Besides, 55 new circulating variants (NCVs) belonging to 41 pangolin lineages were detected. Of these NCVs, 24 were originated throughout the world, and the other 31 in Asturias (10 were only detected in the region, 8 also in Spain, and 13 around the world).Conclusions: New variants circulating around the world continually appear in our region, leading to a constant rotation of lineages. Although most of the changes have little or no impact on the virus's properties, the continued emergence of new variants warrant continued genomic surveillance.

Enteroviruses (EVs) are a large group of genotypes that cause a variety of pathologies, some of them very serious. This study analyzed the last 10 years (2014–2023) of EVs diagnosis and classification. In 166,674 samples collected, EVs were found in 9,535 (5.7%) by rt-RT-PCR, and 332 (3.5%) were classified by Sanger methods. Symptoms were analyzed in 7623 cases. EVs were found in 5718/63,829 (8.9%) before, 1384/42,373 (3.3%) during and 2433/60,472 (4%) after the Covid pandemic (p < 0.0001), and in 7249/69,700 (10.4%) children under 6 years and in 2286/96,974 (2.35%) in oldest (p < 0.0001). The positive rate of EVs was high but decreased during the Covid period. In the youngest children EVs-A (associated with exantematohous disorders as well as respiratory manifestations and febrile syndromes) was most common, while EVs-B (frequent in neurological symptoms) was most common in children aged 6–15 years and EVs-D (associated to respiratory manifestations) in adults.

A lack of HIV viral load (VL) and HIV drug resistance (HIVDR) monitoring in sub-Saharan Africa has led to an uncontrolled circulation of HIV-strains with drug resistance mutations (DRM), compromising antiretroviral therapy (ART). This study updates HIVDR data and HIV-1 variants in Equatorial Guinea (EG), providing the first data on children/adolescents in the country. From 2019–2020, 269 dried blood samples (DBS) were collected in Bata Regional Hospital (EG) from 187 adults (73 ART-naïve/114 ART-treated) and 82 children/adolescents (25 HIV-exposed-ART-naïve/57 ART-treated). HIV-1 infection was confirmed in Madrid by molecular/serological confirmatory tests and ART-failure by VL quantification. HIV-1 pol region was identified as transmitted/acquired DRM, predicted antiretroviral susceptibility (Stanfordv9.0) and HIV-1 variants (phylogeny). HIV infection was confirmed in 88.1% of the individuals and virological failure (VL > 1000 HIV-1-RNA copies/mL) in 84.2/88.9/61.9% of 169 ART-treated children/adolescents/adults. Among the 167 subjects with available data, 24.6% suffered a diagnostic delay. All 125 treated had experienced nucleoside retrotranscriptase inhibitors (NRTI); 95.2% were non-NRTI (NNRTI); 22.4% had experienced integrase inhibitors (INSTI); and 16% had experienced protease inhibitors (PI). At sampling, they had received 1 (37.6%), 2 (32%), 3 (24.8%) or 4 (5.6%) different ART-regimens. Among the 43 treated children–adolescents/37 adults with sequence, 62.8/64.9% carried viruses with major-DRM. Most harbored DRM to NNRTI (68.4/66.7%), NRTI (55.3/43.3%) or NRTI+NNRTI (50/33.3%). One adult and one child carried major-DRM to PI and none carried major-DRM to INSTI. Most participants were susceptible to INI and PI. DRM was absent in 36.2% of treated patients with VL > 1000 cp/mL, suggesting adherence failure. TDR prevalence in 59 ART-naïve adults was high (20.3%). One-half (53.9%) of the 141 subjects with pol sequence carried CRF02_AG. The observed high rate of ART-failure and transmitted/acquired HIVDR could compromise the 95-95-95-UNAIDS targets in EG. Routine VL and resistance monitoring implementation are mandatory for early detection of ART-failure and optimal rescue therapy selection ART regimens based on PI, and INSTI can improve HIV control in EG.

In January 2022, there was a global and rapid surge of the Omicron variant of SARS-CoV-2 related to more transmission. This coincided with an increase in the incidence in Asturias, a region where rapid diagnosis and containment measures had limited the circulation of variants. Methods: From January to June 2022, 34,591 variants were determined by the SNP method. From them, 445 were characterized by the WGS method and classified following pangolin program and phylogenic analysis. Results: The Omicron variant went from being detected in 2438 (78%) samples in the first week of January 2021 to 4074 (98%) in the third week, according to the SNP method. Using the WGS method, 159 BA.1 (35.7%), 256 BA.2 (57.6%), 1 BA.4 (0.2%) and 10 BA.5 (2.2%) Omicron variants were found. Phylogenetic analysis detected that three new sub-clades, BA.2,3.5, BA.2.56 and BF1, were circulating. Conclusions: The increase in the incidence of SARS-CoV2 caused the circulation of new emerging variants. Viral evolution calls for continuous genomic surveillance.

In December 2020, UK authorities warned of the rapid spread of a new SARS-CoV-2 variant, belonging to the B.1.1.7 lineage, known as the Alpha variant. This variant is characterized by 17 mutations and 3 deletions. The deletion 69–70 in the spike protein can be detected by commercial platforms, allowing its real-time spread to be known. From the last days of December 2020 and over 4 months, all respiratory samples with a positive result for SARS-CoV-2 from patients treated in primary care and the emergency department were screened to detect this variant based on the strategy S gene target failure (SGTF). The first cases were detected during week 53 (2020) and reached >90% of all cases during weeks 15–16 (2021). During this period, the B.1.1.7/SGTF variant spread at a rapid and constant replacement rate of around 30–36%. The probability of intensive care unit admission was twice higher among patients infected by the B.1.1.7/SGTF variant, but there were no differences in death rate. During the peak of the third pandemic wave, this variant was not the most prevalent, and it became dominant when this wave was declining. Our results confirm that the B.1.1.7/SGTF variant displaced other SARS-CoV-2 variants in our healthcare area in 4 months. This displacement has led to an increase in the burden of disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in Madrid, Spain, on 25 February 2020. It increased in frequency very fast and by the end of May more than 70,000 cases had been confirmed by reverse transcription-polymerase chain reaction (RT-PCR). To study the lineages and the diversity of the viral population during this first epidemic wave in Madrid we sequenced 224 SARS-CoV-2 viral genomes collected from three hospitals from February to May 2020. All the known major lineages were found in this set of samples, though B.1 and B.1.5 were the most frequent ones, accounting for more than 60% of the sequences. In parallel with the B lineages and sublineages, the D614G mutation in the Spike protein sequence was detected soon after the detection of the first coronavirus disease 19 (COVID-19) case in Madrid and in two weeks became dominant, being found in 80% of the samples and remaining at this level during all the study periods. The lineage composition of the viral population found in Madrid was more similar to the European population than to the publicly available Spanish data, underlining the role of Madrid as a national and international transport hub. In agreement with this, phylodynamic analysis suggested multiple independent entries before the national lockdown and air transportation restrictions.

Background: Phylogenetic analyses of the bacterial genomes based on the simple classification in core- genes and accessory genes pools could offer an incomplete view of the evolutionary processes, of which some are still unresolved. A combined strategy based on stratified phylogeny and ancient molecular polymorphisms is proposed to infer detailed evolutionary reconstructions by using a large number of whole genomes. This strategy, based on the highest number of genomes available in public databases, was evaluated for improving knowledge of the ancient diversification of E. coli. This staggered evolutionary scenario was also used to investigate whether the diversification of the ancient E. coli lineages could be associated with particular lifestyles and adaptive strategies. Results: Phylogenetic reconstructions, exploiting 6220 available genomes in Genbank, established the E. coli core genome in 1023 genes, representing about 20% of the complete genome. The combined strategy using stratified phylogeny plus molecular polymorphisms inferred three ancient lineages (D, EB1A and FGB2). Lineage D was the closest to E. coli root. A staggered diversification could also be proposed in EB1A and FGB2 lineages and the phylogroups into these lineages. Several molecular markers suggest that each lineage had different adaptive trajectories. The analysis of gained and lost genes in the main lineages showed that functions of carbohydrates utilization (uptake of and metabolism) were gained principally in EB1A lineage, whereas loss of environmental-adaptive functions in FGB2 lineage were observed, but this lineage showed higher accumulated mutations and ancient recombination events. The population structure of E. coli was re-evaluated including up to 7561 new sequenced genomes, showing a more complex population structure of E. coli, as a new phylogroup, phylogroup I, was proposed. Conclusions: A staggered reconstruction of E. coli phylogeny is proposed, indicating evolution from three ancestral lineages to reach all main known phylogroups. New phylogroups were confirmed, suggesting an increasingly complex population structure of E. coli. However these new phylogroups represent < 1% of the global E. coli population. A few key evolutionary forces have driven the diversification of the two main E. coli lineages, metabolic flexibility in one of them and colonization-virulence in the other.

The rapidly increasing availability of whole genomes provides the opportunity to reach an updated comprehensive view of bacterial evolution. The staggered diversification of evolutionary processes, based on the combined strategy of layered phylogenomics and polymorphism fingerprinting, give a new perspective in phylogenetic reconstructions. Layered phylogenomics is based on the assignation of genes according to five different evolutionary layers: minimal genome, genus-core genome, species-core genome, phylogroup-core genome and phylogroup-flexible genome. Polymorphism fingerprinting is based on the detection of conserved positions in each phylogenetic group but differing from those of their hypothetical ancestors. This approach was applied to Escherichia coli because there are unresolved evolutionary questions, although has been highly studied. Phylogenetic analysis based on 6,220 full genomes, identified three E. coli root lineages, defined as D, EB1A and FGB2. A new phylogroup, called G was detected near to phylogroup B2. The closest phylogroup to ancestral E. coli was phylogroup D, whereas E and F were the closest ones in their respective lineages; moreover, A and B2 were the most distant phylogroups in EB1A and FGB2 respectively. We suspect that EB1A and FGB2 lineages represent different adaptive strategies. In the deepest branch of EB1A lineage, the number of accumulated mutations was lower than in recent branches, whereas in FGB2 lineage the opposite occurred. The FGB2 lineage was enriched in genes related to host colonization-pathogenicity and toxin-antitoxin systems (such as hipA), whereas B1A sub-lineage acquired functions related to uptake and metabolism of carbohydrates (such as bgl, mng or xlyE). This new combined strategy shows a detailed staggered evolutionary reconstruction, which help us to understand the deepest events and the selection forces have driven E. coli diversification. This approach could add resolution in the reconstruction of the evolutionary trajectories of other microorganisms.