key: cord-0802094-07dy2nrd authors: Mallon, P. W. G.; Crispie, F.; Gonzalez, G.; Tinago, W.; Garcia Leon, A. A.; McCabe, M.; de Barra, E.; Yousif, O.; Lambert, J. S.; Walsh, C. J.; Kenny, J. G.; Feeney, E.; Carr, M.; Doran, P.; Cotter, P. D. title: Whole-genome sequencing of SARS-CoV-2 in the Republic of Ireland during waves 1 and 2 of the pandemic date: 2021-02-10 journal: nan DOI: 10.1101/2021.02.09.21251402 sha: f6fa6b8012c8ddad056ad5bf591fc2663d36200e doc_id: 802094 cord_uid: 07dy2nrd Abstract: Background: Whole-genome sequencing (WGS) of SARS-CoV-2 laboratory-confirmed cases can provide insights into viral transmission and genetic diversity at a population level. However, less is known about the impact of non-pharmaceutical interventions (NPIs), including lockdowns, on circulating SARS-CoV-2 lineages and variants of concern, the relative contribution of travel to re-emergence of pandemic waves within communities or how different lineages and variants contribute to disease severity. Methods: We have conducted an analysis within a prospective, multicentre observational study of individuals attending four hospitals in the South-East of Ireland with COVID-19. Samples underwent WGS from which lineages and variants were assigned, lineage frequency was plotted over time and phylogenetic analysis was employed to determine the origin of variants detected post-lockdown. Univariate and multivariate analyses assessed relationships between viral lineage/variant and COVID-19 disease severity. Results: We analysed 225 genome sequences across two SARS-CoV-2 waves, 134 (59.6%) from wave 1 (March to June) and 91 (40.4%) from wave 2 (July to December), representing 15.2% of COVID-19 admissions to these hospitals during the sampling periods. Four variants (B.1.1.162, B1.1.70, B.1.1.267 and B.1.1) comprised 68% of variants detected during wave 1. Of these variants, only a single B.1.1.70 sequence was detected in wave 2, while the B.1.177 lineage emerged and contributed to 82.3% of lineages detected. Phylogenetic analysis suggested multiple introductions of wave 2 variants from outside Ireland. We found no consistent association between SARS-CoV-2 lineages and disease severity. Conclusions: These data suggest elimination of common SARS-CoV-2 lineages from hospitalised cases associated with effective NPIs and that importation of new viral variants through travel was a significant contributor to the re-emergence of the pandemic in the second wave in Ireland. Our findings highlight the importance of genomic surveillance in identifying circulating viral genetic diversity and variants of concern and, also, modelling the disease burden of SARS-CoV-2. Conclusions: These data suggest elimination of common SARS-CoV-2 lineages from hospitalised cases associated with effective NPIs and that importation of new viral variants through travel was a significant contributor to the re-emergence of the pandemic in the second wave in Ireland. Our findings highlight the importance of genomic surveillance in identifying circulating viral genetic diversity and variants of concern and, also, modelling the disease burden of SARS-CoV-2. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 10, 2021. ; https://doi.org/10.1101/2021.02.09.21251402 doi: medRxiv preprint More than a year after being first reported in Wuhan, Hubei province, China, [1] the COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS- continues to cause significant morbidity and mortality worldwide. In the Republic of Ireland, the first case of community transmission of SARS-CoV-2 was identified on 21st February 2020 [2] . Since then, at the time of writing (Feb 7, 2021), over 201,763 cases have been reported, linked to 3,621 deaths (www.hpsc.ie). The pandemic in Ireland has been characterised by three distinct 'waves' of infections, each controlled by non-pharmaceutical interventions (NPIs) involving a series of societal restrictions comprising physical distancing, hand hygiene and limiting movements within the country. During the first wave, implementation of these restrictions (or 'lockdowns') was associated with significant reductions in daily reported cases and mortality, followed by phased relaxation in restrictions [3] . Despite easing of restrictions on 18th May, this impact was maintained, with lowest daily reported case numbers of 3 laboratory-confirmed cases reported at the end of June 2020 [4] . This was achieved while permitting international travel through the country's airports and seaports and permitting travel across the land border between the Republic of Ireland and Northern Ireland. Following phase three easing of restrictions in late June, which included permission to travel within Ireland, case numbers began to rise and subsequent efforts at control of transmission through community restrictions have failed to achieve the effects seen in June 2020. The highest (level 5) restrictions were implemented on 21st October 2020 with the lowest subsequent reported case numbers at the end of 'wave 2' dropping to 183 on 3rd December 2020, at the time when level 5 restrictions began to be relaxed. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Since first being reported in China, the evolution of the virus through mutations has enabled epidemiological mapping of the virus through phylogenetic analyses of viral genome sequences. This genomic epidemiology provides invaluable insights into the origins of viral transmissions within countries, and the impact of the mutations on either transmission or disease severity. For example, the identification of the 20AEU1 or B.1.177 variant, first identified in Spain on June 20 th , 2020, was subsequently reported across Europe [5] . Although not linked to increased transmission or mortality, its increased prominence in the subsequent months across Europe supported holiday-related travel as an important factor in its spread. Since then, additional variants of concern, such as the B.1.1.7, B.1.351, and P.1 variants have been identified, [6, 7] which have been linked to both increased transmissibility and potentially worse clinical outcomes [8] . Although a number of studies have described genotypic variation in SARS-CoV-2 across Europe, few have been able to track the impact of effective NPIs, such as an effective lockdown as occurred during the first wave in Ireland, on viral genetic diversity in a representative sample of reported cases, and few studies have related differences in viral lineage to clinical outcomes [9] . Our objectives in the present study were to describe the genetic variation in SARS-CoV-2 lineages among individuals hospitalised with COVID-19 in the East and South-East of Ireland during 2020, covering two of the three 'waves' of the SARS-CoV-2 pandemic in Ireland, to define the impact of lockdown on lineage diversity and to explore the geographical source of infections during the course of the pandemic. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Following sequencing, Fast5 files were uploaded to the Irish Centre for High End Computing All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 10, 2021. ; https://doi.org/10.1101/2021.02.09.21251402 doi: medRxiv preprint (ICHEC) and bioinformatics analysis including genome assembly and variant calling were performed as described by the ARTIC network [12] . FastA files of SARS-CoV2 whole genome consensus sequences were used for assignment of a Pangolin lineage to each sample. The PANGOLIN nomenclature offers an algorithmic method to classify, distinguish and put in context the different SARS-CoV-2 sequences considering the large amount of available genomic sequence data [13] in public databases, such as GISAID (www.gisaid.org) [14] . However, as the characterisation of the different SARS-CoV-2 lineages become available and more sequences are accumulated retrospectively, some of these lineages are revised and reassigned [15] . Consequently, the following descriptions are subject to revisions to such a dynamic nomenclature. The lineages assigned in this report were based on the pangoLEARN version 2021/02/01. For reference, the characteristic non-synonymous mutations in the SARS-CoV-2 spike protein associated with these lineages are provided in Table 1 . Lineages were assigned to samples, aligned with metadata from the AIID Cohort (age, gender and county of residence), and genome sequences were uploaded onto the public database GISAID. Global genomic sequences were downloaded from GISAID [14] . Of these genomes, those with two or less nucleotide differences to any of the Irish B.1.177 sequences were selected. Subsequently, 310 sequences with the earliest sampling dates were chosen and multiple sequences aligned to the reference (MN908947) and 71 AIID B.1.177 genomic sequences with MAFFT [16] . A phylogenetic tree was inferred from this multiple sequence alignment All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 10, 2021. ; https://doi.org/10.1101/2021.02.09.21251402 doi: medRxiv preprint with RAxML [17] with the general time reversible model of substitution allowing for heterogeneity among sites (GTRCAT). We used summary statistics as appropriate and bubble plots to describe the patterns of distribution of genome lineages over time (month or week from 24 th March to 27 th December 2020). Bubble charts were created in R with ggplot2 packages [18] . We used ordinal logistic regression (proportional odds model) to examine the associations between the most frequently identified lineages and a primary outcome measure of maximal COVID-19 disease severity defined according to the WHO guidance [19] and collapsed into mild, moderate and severe for analysis. The cumulative odds ratios (OR) for the proportional odds logistic regression represented the cumulative odds of a more severe clinical disease ("Severe vs Moderate/Mild disease, or "Severe/Moderate vs Mild disease). The proportional odds assumption were tested using the chi-square score test and graphical assessments to check for parallelism. All statistical analyses were performed in SAS version 9.4 (SAS Institute), and R version 4.0.2 (Vienna, Austria) [20] . Between 24 th March and 27th December 2020, 1,211 subjects were recruited to the AIID Cohort, of which 843 (69.6%) were laboratory-confirmed SARS-CoV-2 positive by qRT-PCR. Of these, 275 subjects from four participating hospitals provided 660 respiratory samples for biobanking of which 225 were suitable for genome sequencing. Demographics of participating subjects broadly reflected the demographics of hospitalised cases of COVID-19 in Ireland (Table 2) , with an average age of 65 years, predominantly male (55%) and All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 10, 2021. ; Caucasian (83%). Greater than 80% of subjects reported an underlying condition, with hypertension, chronic respiratory disease, chronic heart disease and malignancy being the most common underlying conditions reported. A substantial portion of subjects experienced either moderately severe or severe COVID-19 infection, with an overall 10.6% mortality rate. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Phylogenetic analysis of B.1.177 genomes from our analysis, when compared to genome sequences available in GISAID that had less than two mutations separating from our cases, supported multiple introductions of this lineage to Ireland from different European countries All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 10, 2021. identified as English cases. In addition, monophyletic clusters of Irish samples also reflect some divergence accumulated through community transmission. Overall, these data suggest multiple sources of origin, consistent with multiple, distinct introductions of these variants into Ireland (Figure 4) , likely through travel. Although the B.1.1.7 variant of concern, first reported in Kent in the UK from samples taken in late September 2020 [21] (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This is the first study to describe the change in lineages of SARS-CoV-2 in Ireland in 2020 from a representative sample of cases linked to clinically relevant data. Our analyses reveal three main findings; that, compared to the second wave, the first SARS-CoV-2 wave in Ireland was characterised by a larger number of distinct lineages, that the use of successful NPIs in the form of an effective 'lockdown' was accompanied by the disappearance of these lineages from those contributing to hospitalisations among the participating hospitals, and that the second wave was derived from viral genetic lineages that were most closely phylogenetically linked to genotypes outside of Ireland, suggesting multiple introductions through travel during the summer of 2020. We observed distinct lineages of SARS-CoV-2 contributing to wave 1 and wave 2 of (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 10, 2021. ; https://doi.org/10.1101/2021.02.09.21251402 doi: medRxiv preprint a United Kingdom lineage [15] . These data suggest that commonly reported lineages during wave 1 contributing to hospitalisations in our cohort included a mixture of originally imported lineages from continental Europe along with viruses that had evolved within were detected predominantly in our hospitalised cohort, representing 82.4% samples analysed from the second wave. This lineage has been traced to Spain in early March; however, it became widespread across Europe during July and August 2020 [5] . Our phylogenetic analysis points to multiple introductions of this lineage into Ireland from overseas, in particular from the United Kingdom, with smaller clusters within Irish samples also reflecting continued divergence of this lineage through onward community transmission as it became established in Ireland. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 10, 2021. ; https://doi.org/10.1101/2021.02.09.21251402 doi: medRxiv preprint Taken together, these data suggest that the first lockdown may have largely eradicated these commonly reported lineages from the circulating pool of transmitting viruses contributing to hospitalisations in the south-east Ireland, and that the second wave of infections resulting in hospitalisations were largely seeded by multiple, travel-related events from countries outside of the island of Ireland. The importance of travel contributing to changes in lineages and variants of concern is further underlined by the detection of the B. Investigation, year 2020, month 12, variant 01), has grown in frequency in the United Kingdom becoming the most prevalent lineage with a similar trend also observed in other European countries, including Ireland and Denmark [28] . Members of this lineage are characterised by a higher number of characteristic, non-synonymous, amino acid mutations in the spike protein ( Table 1 ) that have been hypothesized to have originated from virus evolution in a chronically-infected individual [21] . Among the mutations described in the B.1.1.7 variant, the N501Y mutation, localised in the receptor binding site has been shown to increase the binding of SARS-CoV-2 to the angiotensin-converting enzyme 2 (ACE2) host receptor [29] , inferring increased viral fitness and transmission properties. Although some data suggest that this variant may escape the neutralisation by select convalescent sera and therapeutic monoclonal antibodies, sera from participants of a trial of the mRNA based COVID vaccine BNT162b2 neutralised with All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 10, 2021. ; 1 6 similar efficacy the wild type N501 and the variant N501Y [30] suggesting retained vaccine efficacy against the B.1.1.7 variant. We explored associations between presence of specific SARS-CoV-2 lineages and maximal disease severity experienced by cohort participants, according to WHO classification [19] . Data on relationships between SARS-CoV-2 lineages and disease severity are lacking; we could find no prior reports analysing relationships between B.1.1.269 and COVID-19 disease severity. Although univariate analyses did suggest that the B.1.177 lineage was associated with a significantly reduced odds of either moderate or severe COVID-19, these associations were prone to multiple bias, including differences in the demographic of patients presenting during wave 1 and wave 2, as well as the introduction of more standardised approaches to patient management in the second wave, including increased use of treatments such as remdesivir and dexamethasone [31] [32] [33] . Additionally, the associations were significantly abrogated by adjustment for patient demographics and overall our analysis does not support any robust relationship between the more frequently observed variants and disease severity. outbreaks or to detect variants of interest. However, as the AIID cohort took an unselected approach to sampling and did not pre-screen for variants of interest, the impact of any sampling bias would have been less evident. In conclusion, we have described disappearances of viral lineages from hospitalised COVID-19 cases during wave 1 of the pandemic in Ireland occurring alongside lockdown restrictions, and emergence of different SARS-CoV-2 lineages contributing to hospitalisations during wave 2, with phylogenetic analysis pointing to importation through travel as an important source. These findings show the utility of SARS-CoV-2 genomic epidemiology in providing insights into viral genetic lineages and variants of concern and have significant implications for reflective learning as part of pandemic control of SARS-CoV-2 infection. Whole genome sequencing of SARS-CoV-2 isolates provides critical information for the monitoring for the emergence of new viral lineages and variants of concern. 1 8 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 10, 2021. ; https://doi.org/10.1101/2021.02.09.21251402 doi: medRxiv preprint The size of the circle is proportional to the number of lineages detected. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 10, 2021. ; https://doi.org/10.1101/2021.02.09.21251402 doi: medRxiv preprint The size of the circle is proportional to the number of lineages detected. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 10, 2021. ; https://doi.org/10.1101/2021.02.09.21251402 doi: medRxiv preprint The size of the circle is proportional to the number of lineages detected. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 10, 2021. ; https://doi.org/10.1101/2021.02.09.21251402 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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