Dispersal history of SARS-CoV-2 in Galicia, Spain (preprint)

The dynamics of SARS-CoV-2 transmission are influenced by a variety of factors, including social restrictions and the emergence of distinct variants. In this study, we delve into the origins and dissemination of the Alpha, Delta, and Omicron variants of concern in Galicia, northwest Spain. For this, we leveraged genomic data collected by the EPICOVIGAL Consortium and from the GISAID database, along with mobility information from other Spanish regions and foreign countries. Our analysis indicates that initial introductions during the Alpha phase were predominantly from other Spanish regions and France. However, as the pandemic progressed, introductions from Portugal and the USA became increasingly significant. Notably, Galicia's major coastal cities emerged as critical hubs for viral transmission, highlighting their role in sustaining and spreading the virus. This research emphasizes the critical role of regional connectivity in the spread of SARS-CoV-2 and offers essential insights for enhancing public health strategies and surveillance measures.

Adaptive advantage of deletion repair in the N terminal domain of the SARS-CoV-2 spike protein in variants of concern (preprint)

Mutations within the N-terminal domain (NTD) of the spike (S) protein play a pivotal role in the emergence of successful SARS-CoV-2 viral lineages. This study investigates the influence of novel combinations of NTD lineage-defining mutations found in the Alpha, Delta, and Omicron variants on viral success. We performed comparative genomics of more than 10 million public SARS-CoV-2 samples to decipher the transmission success of different NTD markers. Additionally, we characterized the viral phenotype of such markers in a surrogate in vitro system. We found that viruses bearing repaired deletions SDeltaH69/V70 and SDeltaY144 in Alpha background were associated with increased transmission rates. After the emergence of the Omicron BA.1 lineage, Alpha viruses harbouring both repaired deletions still showed increased transmission compared to their BA.1 counterparts. Remarkably, Alpha viruses with the SDeltaH69/V70 repair displayed the highest emergence rate, while those in BA.1 exhibited the lowest. Moreover, repaired deletions were more frequently observed among older individuals infected with Alpha, but not with BA.1. In vitro biological characterization of Omicron BA.1 spike deletion repair patterns revealed substantial differences with Alpha. In BA.1, SDeltaV143/Y145 repair enhanced fusogenicity and susceptibility to neutralization by vaccinated individuals' sera. In contrast, the SDeltaH69/V70 repair did not significantly alter these traits but reduced viral infectivity. Simultaneous repair of both deletions led to lower fusogenicity. These findings highlight the intricate genotype-phenotype landscape of the spike NTD in SARS-CoV-2, which impacts viral biology, transmission efficiency, and susceptibility to neutralization. Overall, this study advances our comprehension of SARS-CoV-2 evolution, carrying implications for public health and future research.

VIPERA: Viral Intra-Patient Evolution Reporting and Analysis (preprint)

Viral mutations within patients nurture the adaptive potential of SARS-CoV-2 during chronic infections, which are a potential source of variants of concern. However, there is no integrated framework for the evolutionary analysis of intra-patient SARS-CoV-2 serial samples. Herein we describe VIPERA (Viral Intra-Patient Evolution Reporting and Analysis), a new software that integrates the evaluation of the intra-patient ancestry of SARS-CoV-2 sequences with the analysis of evolutionary trajectories of serial sequences from the same viral infection. It has been validated using positive and negative control datasets and successfully applied to a novel case, contributing to easy and automatic analysis of intra-patient SARS-CoV-2 sequences.

Circulation of SARS-CoV-2 and co-infection with Plasmodium falciparum in Equatorial Guinea (preprint)

The impact of COVID-19 in Africa has been a big concern since the beginning of the pandemic. However, low incidence of COVID-19 case severity and mortality has been reported in many African countries, although data are highly heterogeneous and, in some regions, like Sub-Saharan Africa, very scarce. Many of these regions are also the cradle of endemic infectious diseases like malaria. The aim of this study was to determine the prevalence of SARS-CoV-2, the diversity and origin of circulating variants as well as the frequency of co-infections with malaria in Equatorial Guinea. For this purpose, we conducted antigen diagnostic tests for SARS-CoV-2, and microscopy examinations for malaria of 1,556 volunteers at six health centres in Bioko and Bata from June to October 2021. Nasopharyngeal swab samples were also taken for molecular detection of SARS-CoV-2 by RT-qPCR and whole genome viral sequencing. We report 3.0% of SARS-CoV-2 and 24.4% of malaria prevalence over the sampling in Equatorial Guinea. SARS-CoV-2 cases were found at a similar frequency in all age groups, whereas the age groups most frequently affected by malaria were children (36.8% [95% CI 30.9-42.7]) and teenagers (34.7% [95% CI 29.5-39.9]). We found six cases of confirmed co-infection of malaria and SARS-CoV-2 distributed among all age groups, representing a 0.4% frequency of co-infection in the whole sampled population. Interestingly, the majority of malaria and SARS-CoV-2 co-infections were mild. We obtained the genome sequences of 43 SARS-CoV-2 isolates, most of which belong to the lineage Delta (AY.43) and that according to our pandemic-scale phylogenies were introduced from Europe in multiple occasions (7 transmission groups and 17 unique introductions). This study is relevant in providing first-time estimates of the actual prevalence of SARS-CoV-2 in this malaria-endemic country, with the identification of circulating variants, their origin, and the occurrence of SARS-CoV-2 and malaria co-infection.

Prevalence of SARS-CoV-2 and co-occurrence/co-infection with malaria during the first wave of the pandemic (the Burkina Faso case) (preprint)

Africa accounts for 1.5% of the global coronavirus disease 2019 (COVID-19) cases and 2.7% of deaths, but this low incidence has been partly attributed to the limited testing capacity in most countries. In addition, the population in many African countries is at high risk of infection with endemic infectious diseases such as malaria. Our aim is to determine the prevalence and circulation of SARS-CoV-2 variants, and the frequency of co-infection with the malaria parasite. We conducted serological tests and microscopy examinations on 998 volunteers of different ages and sexes in a random and stratified population sample in Burkina-Faso. In addition, nasopharyngeal samples were taken for RT-qPCR of SARS-COV-2 and for whole viral genome sequencing. Our results show a 3.2% and a 2.5% of SARS-CoV-2 seroprevalence and PCR positivity; and 22% of malaria incidence, over the sampling period, with marked differences linked to age. Importantly, we found 2 cases of confirmed co-infection and 8 cases of suspected co-infection mostly in children and teenagers. Finally, we report the genome sequences of 13 SARS-CoV-2 isolates circulating in Burkina Faso at the time of analysis, assigned to lineages: A.19, A.21, B.1.1.404, B.1.1.118, B.1 and grouped into clades; 19B, 20A and 20B. This is the first population-based study about SARS-CoV-2 and malaria in Burkina Faso during the first wave of the pandemic, providing a relevant estimation of the real prevalence of SARS-CoV-2 and variants circulating in this Sub-Saharan African country. Besides, it highlights the low frequency of co-infection with malaria in African communities.

Cumulative incidence of SARS-CoV-2 infection in the general population of the Valencian Community (Spain) after the surge of the Omicron BA.1 variant (preprint)

Background Studies investigating the cumulative incidence of and immune status against SARS-CoV-2 infection provide valuable information for shaping public health decision- making. Methods The current cross-sectional, population-based study, conducted in April 2022 in the Valencian Community (VC), recruited 935 participants of all ages. Anti-SARS-CoV-2-Receptor Binding Domain-RBD- total antibodies and anti-Nucleocapsid (N)- IgGs were measured by electrochemiluminescence assays. To account for past SARS-CoV-2 infection the VC microbiology registry (RedMiVa) was interrogated. Quantitation of neutralizing antibodies (NtAb) against the ancestral and Omicron BA.1 and BA.2 (sub)variants by an S-pseudotyped neutralization assay and for enumeration of SARS-CoV-2-S specific-IFNgamma;-producing CD4+ and CD8+ T cells by Intracellular Cytokine Staining assay was performed in a subset of participants (n=100 and 137, respectively). Findings The weighted cumulative incidence was 51.9% (95% CI, 48.7-55.1), and was inversely related to age. Anti-RBD total antibodies were detected in 906/931 (97.3%) participants, those vaccinated and SARS-CoV-2-experienced (VAC-ex;=442) displaying higher levels (P<0.001) than vaccinated/naive (VAC-n;(n=472) and non-vaccinated/experienced (UNVAC-ex; n(n=63). Antibody levels correlated inversely with the time elapsed since receipt of last vaccine dose in VAC-n (Rho, -0.52; 95% CI, -0.59 to -0.45; P<0.001) but not in VAC-ex. NtAbs against Omicron BA.1 were detected in 94%, 75% and 50% of VAC-ex, VAC-n and UNVAC-ex groups, respectively, while in 97%, 84% and 40%, against Omicron BA.2. SARS-CoV-2-S-reactive IFNgamma; T cells were detected in 73%, 75%, and 64% for VAC-ex, VAC-n, UNVAC-ex, respectively. Interpretation By April 2022 around half of the VC population had been infected with SARS-CoV-2 and due to extensive vaccination display hybrid immunity. The large percentage of participants with detectable functional antibody and T-cell responses against SARS-CoV-2, which may be cross-reactive to some extent, points towards lower expected severity than in previous waves.

Deciphering the tangible spatio-temporal spread of a 25 years tuberculosis outbreak boosted by social determinants (preprint)

Background Outbreak strains are good candidates to look for intrinsic transmissibility as they are responsible for a large number of cases with sustained transmission. However, assessment of the success of long-lived outbreak strains has been flawed by the use of low-resolution typing methods and restricted geographical investigations. We now have the potential to address the nature of outbreak strains by combining large genomic datasets and phylodynamic approaches. Methods We retrospectively sequenced the whole genome of representative samples assigned to an outbreak circulating in the Canary Islands (GC) since 1993; accounting for ∼20% of local TB cases. We selected a panel of specific SNP markers to in-silico search for additional outbreak related sequences within publicly-available TB genomic data. Using this information we inferred the origin, spread and epidemiological parameters of the GC-outbreak. Findings Our approach allowed us to accurately trace both the historical and recent dispersion of the strain. We evidenced its high success within the Canarian archipelago but found a limited expansion abroad. Estimation of epidemiological parameters from genomic data contradicts a distinct biology of the GC-strain. Interpretation With the increasing availability of genomic data allowing for an accurate inference of strain spread and key epidemiological parameters, we can now revisit the link between Mycobacterium tuberculosis genotypes and transmission, as routinely done for SARS-CoV-2 variants of concern. We show that the success of the GC-strain is better explained by social determinants rather than intrinsically higher bacterial transmissibility. Our approach can be used to trace and characterize strains of interest worldwide. Funding European Research Council (101001038-TB-RECONNECT), the Ministerio de Economía, Industria y Competitividad (PID2019-104477RB-I00), Instituto de Salud Carlos III (FIS18/0336), European Commission –NextGenerationEU (Regulation EU 2020/2094), through CSIC’s Global Health Platform (PTI Salud Global) to IC. Gobierno de Aragón/Fondo Social Europeo “Construyendo Europa desde Aragón” to SS Research in context Evidence before this study Identification of intrinsically highly transmissible strains of Mycobacterium tuberculosis remains elusive. Among candidates are those strains that have been thriving in a community for decades representing a significant contribution to the long-term local TB burden. These long-lived outbreak strains have been identified in different parts of the world and the speculation is that their success is linked to higher transmissibility. Several studies have attempted to analyze the epidemiological characteristics of these strains as well as their genomic composition to look for potential transmission determinants. However those studies are usually circunscribe to their original geographic boundaries. By contrast, this transmissibility should be replicated in different parts of the world, a lesson learnt from SARS-CoV-2 variants of concern. Previous attempts failed to examine the success of these outbreak strains at a global scale. Thus, it is unknown whether the long-lived outbreak strains had a similar or different trajectory in other countries, casting doubts about their transmissibility potential. Added value of this study Here we analyzed a strain causing a long-lived outbreak in the Canary Islands since 1993 using whole genome sequencing. As in previous studies with other similar outbreak strains, we analyzed the diversity and phylodynamics of the outbreak in the area where it was originally described. However, thanks to the possibility of interrogating the entire European Nucleotide Archive, we had the unique chance to look at the spread of the strains beyond its original geographic boundaries. This approach allowed us to comprehensively trace the real spatio-temporal spread of the outbreak from the emergence of its ancestor about 700 years ago to its recent transmission outside the Canary Islands. As a result, there is limited evidence for similar success of the strains outside Canary Islands. Furthermore, we complemented the analysis with epidemiological data of the early cases and with phylodynamic analysis to estimate key epidemiological parameters linked to the strain spread. All evidence strongly suggests that factors related to the host, instead of the bacteria, are behind the persistence and expansion of the outbreak strain. Implications of all the available evidence Infectious disease outbreaks are a major problem for public health. Tracing outbreak expansion and knowing the main factors behind their emergence and persistence are key to an effective disease control. Our study allows researchers and public health authorities to use WGS-based methods to trace outbreaks, and include available epidemiological information to evaluate the factors underpinning outbreak persistence. Taking advantage of all the information freely available in public repositories, researchers can accurately establish the expansion of the outbreak behind its original boundaries; and they can determine the potential risk of the strain to inform health authorities which, in turn, can define target strategies to mitigate its expansion and persistence. Finally, we show the need to evaluate strain transmissibility in different geographic contexts to unequivocally associate its spread to local or pathogen factors, a major lesson taken from SARS-CoV-2 genomic surveillance.

The structural role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: the case of the spike A222V mutation (preprint)

The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has now reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness.

Overlapping of independent SARS-CoV-2 nosocomial transmissions in a complex outbreak (preprint)

SARS-CoV-2 nosocomial outbreaks in the first COVID-19 wave were likely associated to a shortage of personal protective equipment and scare indications on control measures. Having covered these limitations, updates on current SARS-CoV-2 nosocomial outbreaks are required. We carried out an in-depth analysis of a 27-day nosocomial outbreak in a gastroenterology ward in our hospital, potentially involving 15 patients and three healthcare workers. Patients had stayed in one of three neighbouring rooms in the ward. The severity of the infections in six of the cases and a high fatality rate suggested the possible involvement of a single virulent strain persisting in those rooms. Whole genome sequencing of the strains from 12 patients and one healthcare worker revealed an unexpected complexity. Five different SARS-CoV-2 strains were identified, two infecting a single patient each, ruling out their relationship with the outbreak; the remaining three strains were involved in three independent overlapping limited transmission clusters with three,  three, and five cases. Whole genome sequencing was key to understand the complexity of this outbreak.

Detection Of Genomic Variants Of SARS-CoV-2 Circulating In Wastewater By High-Throughput Sequencing (preprint)

The use of SARS-CoV-2 metagenomics in wastewater can allow the detection of variants circulating at community level. After comparing with clinical databases, we identified three novel variants in the spike gene, and six new variants in the spike detected for the first time in Spain. We finally support the hypothesis that this approach allows the identification of unknown SARS-CoV-2 variants or detected at only low frequencies in clinical genomes.

The first wave of the Spanish COVID-19 epidemic was associated with early introductions and fast spread of a dominating genetic variant (preprint)

The COVID-19 pandemic has shaken the world since the beginning of 2020. Spain is among the European countries with the highest incidence of the disease during the first pandemic wave. We established a multidisciplinar consortium to monitor and study the evolution of the epidemic, with the aim of contributing to decision making and stopping rapid spreading across the country. We present the results for 2170 sequences from the first wave of the SARS-Cov-2 epidemic in Spain and representing 12% of diagnosed cases until 14th March. This effort allows us to document at least 500 initial introductions, between early February-March from multiple international sources. Importantly, we document the early raise of two dominant genetic variants in Spain (Spanish Epidemic Clades), named SEC7 and SEC8, likely amplified by superspreading events. In sharp contrast to other non-Asian countries those two variants were closely related to the initial variants of SARS-CoV-2 described in Asia and represented 40% of the genome sequences analyzed. The two dominant SECs were widely spread across the country compared to other genetic variants with SEC8 reaching a 60% prevalence just before the lockdown. Employing Bayesian phylodynamic analysis, we inferred a reduction in the effective reproductive number of these two SECs from around 2.5 to below 0.5 after the implementation of strict public-health interventions in mid March. The effects of lockdown on the genetic variants of the virus are reflected in the general replacement of preexisting SECs by a new variant at the beginning of the summer season. Our results reveal a significant difference in the genetic makeup of the epidemic in Spain and support the effectiveness of lockdown measures in controlling virus spread even for the most successful genetic variants. Finally, earlier control of SEC7 and particularly SEC8 might have reduced the incidence and impact of COVID-19 in our country.

A complete analysis of the epidemiological scenario around a SARS-CoV-2 reinfection: previous infection events and subsequent transmission (preprint)

The first descriptions of reinfection by SARS-CoV-2 have been recently reported. However, these studies focus exclusively on the reinfected case, without considering the epidemiological context of the event. We present the first complete analysis of the epidemiological scenario around a reinfection by SARS-CoV-2, including three cases preceding the reinfection, the reinfected case per se, and the subsequent transmission to another seven cases. Our analysis is supported by host genetics, viral whole genome sequencing, phylogenomic population analysis, and refined epidemiological data obtained from in-depth interviews with the involved subjects. The reinfection involved a 53-year-old woman with asthma, with a first COVID-19 episode in April 2020 and a much more severe second episode four months and a half later, with COVID-19 seroconversion in August, and requiring hospital admission.

Emergence and spread of a SARS-CoV-2 variant through Europe in the summer of 2020 (preprint)

A variant of SARS-CoV-2 emerged in early summer 2020, presumably in Spain, and has since spread to multiple European countries. The variant was first observed in Spain in June and has been at frequencies above 40% since July. Outside of Spain, the frequency of this variant has increased from very low values prior to 15th July to 40-70% in Switzerland, Ireland, and the United Kingdom in September. It is also prevalent in Norway, Latvia, the Netherlands, and France. Little can be said about other European countries because few recent sequences are available. Sequences in this cluster (20A.EU1) differ from ancestral sequences at 6 or more positions, including the mutation A222V in the spike protein and A220V in the nucleoprotein. We show that this variant was exported from Spain to other European countries multiple times and that much of the diversity of this cluster in Spain is observed across Europe. It is currently unclear whether this variant is spreading because of a transmission advantage of the virus or whether high incidence in Spain followed by dissemination through tourists is sufficient to explain the rapid rise in multiple countries.