Local occurrence and fast spread of B.1.1.7 lineage: A glimpse into Friuli Venezia Giulia.

In-depth study of the entire SARS-CoV-2 genome has uncovered many mutations, which have replaced the lineage that characterized the first wave of infections all around the world. In December 2020, the outbreak of variant of concern (VOC) 202012/01 (lineage B.1.1.7) in the United Kingdom defined a turning point during the pandemic, immediately posing a worldwide threat on the Covid-19 vaccination campaign. Here, we reported the evolution of B.1.1.7 lineage-related infections, analyzing samples collected from January 1st 2021, until April 15th 2021, in Friuli Venezia Giulia, a northeastern region of Italy. A cohort of 1508 nasopharyngeal swabs was analyzed by High Resolution Melting (HRM) and 479 randomly selected samples underwent Next Generation Sequencing analysis (NGS), uncovering a steady and continuous accumulation of B.1.1.7 lineage-related specimens, joined by sporadic cases of other known lineages (i.e. harboring the Spike glycoprotein p.E484K mutation). All the SARS-CoV-2 genome has been analyzed in order to highlight all the rare mutations that may eventually result in a new variant of interest. This work suggests that a thorough monitoring of the SARS-CoV-2 genome by NGS is essential to contain any new variant that could jeopardize all the efforts that have been made so far to resolve the emergence of the pandemic.

Introduction

The emergence and continuous spread of SARS-CoV-2 viral infections has seriously tested public health infrastructures, and led to millions of infections and deaths worldwide [1]. Over a year into the pandemic, sequencing analysis has shown that SARS-CoV-2 variants are being selected as the ongoing uncontrolled spreading of the SARS-CoV-2. While many mutations represent neutral genetic drift [2], a subset may alter its pathogenic potential and possibly reduce the efficacy of drugs and of immunity elicited by current vaccines. Since isolated, complete viral genome sequences have been periodically uploaded into the Global Initiative on Sharing Avian Influenza Data (GISAID; https://www.gisaid.org) database. GISAID initiated a naming system based on large clades that have been identified through variations from the reference genome. GISAID-deposited genomes have been analyzed in different open-source project among which NextStrain has gained a well-deserved popularity (www.nextstrain.org). It interactively visualizes the phylogeny of an emerging viral pathogen to better understand its dispersion dynamic and to improve outbreak responses. Furthermore, it named evolutionary stable lineages based on the year of emergence and a letter (e.g., Nextstrain Clade: 19A, 19B, 20A, 20B and so on) (https://nextstrain.org/ncov/gisaid/global?l=radial; version v2.29.1), despite some sub lineages have been identified with additional information (e.g., 20E/EU1) but with no systematic rule. Indeed, in mid-2020 Rambaut and colleagues have suggested a third dynamic nomenclature, based on evolutionary relationships and epidemiological relevance and an algorithm named PANGOLIN (Phylogenetic Assignment of Named Global Outbreak Lineages) has been proposed and used to assign lineages to FASTA sequences [3]. Recently, to assist with public discussions of variants, the World Health Organization (WHO) introduced a non-stigmatizing label for SARS-CoV-2 variants using letters of the Greek Alphabet to aid the non-scientific audience. SARS-CoV-2 nomenclature methods have been revised by Alm and colleagues [4] and have been further summarized in S1 Table.

As infections continue to spread, viral genome sequencing coupled to epidemiological surveillance became mandatory to actively monitor all the SARS-CoV-2 variants that are globally circulating and rapidly determine the ongoing virus evolution. Based on this premises, several studies highlighted that the main circulating lineage in Italy were B.1 and B.1.177, together with their sub-lineages, representing about 70% of cases in late 2020 [5-7]. Later on, several variants of concern (VOC), each represented by a constellation of specific mutations selected to enhance viral fitness, have emerged [8]. In December 2020, a new variant was identified in the United Kingdom, firstly named as VOC 202012/01, and representing the PANGO B.1.1.7 lineage. This variant has been recently named Alpha by the WHO classification. It has an estimated 43–90% higher transmissibility than pre-existing lineages, thus causing an increasing concerns on Covid-19 infection rate [9]. In the same period, the VOC 202012/02 variant was first isolated in South Africa, which is associated the B.1.351 lineage, newly named Beta. Finally, in January 2021 a third VOC has been firstly identified in Brazil and then in Japan, named VOC 202101/02, belonging to the P.1 lineage, newly named Gamma. All these lineages were concerning due to their increased transmissibility and putative disease severity.

In this study, we aimed to track and evaluate the diffusion of the different SARS-CoV-2 lineages in the northeastern part of the Italian peninsula, in a cohort of nasopharyngeal swabs collected between January and April 2021.

Materials and methods

Samples’ collection and RNA extraction

SARS-CoV-2 samples were collected from all the nasopharyngeal swabs resulted positive to the Allplex COVID-19 assay on Seegene’s automated system between January 1st, 2021 and April 15th, 2021, in the Department of Laboratory Medicine of the University Hospital of Udine (Italy). SARS-CoV-2 positive samples were randomly selected and tested with primers targeting the E gene, which has been previously demonstrated in our reports to be the most sensitive target [10, 11]. Samples with Ct value ≤ 28 were chosen for further analyses. For swab collection, transportation, and long-term storage UTM® tubes (COPAN Diagnostics) were used. Ethical approval was obtained from the Medical Research Ethics Committee of the Region Friuli Venezia Giulia, Italy (Consent CEUR-2020-Os-033).

Total nucleic acids were extracted from 500 μL UTM-medium on the QIAsymphony SP using the QIAsymphony DSP Virus/Pathogen Midi Kit (Qiagen), following manufacturer’s instructions. Samples were eluted in 60μL AVE buffer and used as template for downstream analysis.

High resolution melting (HRM)

High resolution melting was used for the qualitative detection of p.N501Y and p.E484K mutations in the SARS-CoV-2 genome. Briefly, extracted RNA was used as a template for the VirSNiP SARS B1351 assay on the LightCycler ® 480 Real-Time PCR System (Roche Diagnostics). Positive and negative control (NTC) were included in all experiments. 4 μL Roche master mix, 0.5 μL reagent mix (containing primers and probes), 5.5 μL PCR-grade water and 10 μL of template were mixed to a total volume of 20 μL. Amplification was performed following manufacturer’s instruction. After normalization and temperature shift determination, the different melting curves were generated. p.E484K is associated with a shift from 52°C to 57°C while p.N501Y is associated with a shift from 60°C to 65°C. LightCycler® 480 Software was used to analyze all data.

Library preparation and next-generation sequencing (NGS)

Samples were randomly selected from the 1508 specimens that underwent HRM screening. 479 barcoded libraries were prepared using the Ion AmpliSeq SARS-CoV-2 Research Panel (Thermo Fisher Scientific) and the Ion AmpliSeq Library Kit Plus (Thermo Fisher Scientific), following manufacturer’s protocol. The panel consists of 237 amplicons that allows for the sequencing of 99% of the SARS-CoV-2 reference genome (NC_045512.2), covering from position 43 to position 29,842. All the reactions were performed in a Veriti™Dx 96-Well Thermal Cycler (Applied Biosystems™). Libraries were quantified with the Qubit dsDNA HS Assay kit (Life Technologies) and then diluted to 30 pM. Libraries were loaded into the Ion Chef™ Instrument (Thermo Fisher Scientific) for template enrichment and chip loading. Sequencing was performed with the Ion S5 GeneStudio Sequencer using the Ion 510 & Ion 520 & Ion 530 Kit-Chef and the Ion 530™ chip-kit (all Thermo Fisher Scientific).

Data analysis and variant prioritization

After alignment, the following plugins were run on the Torrent Suite™ Server (Thermo Fisher Scientific): the SARS_CoV_2_variant caller, the SARS_CoV_2_CoverageAnalysis, the SARS_CoV_2_annotateSnpEff and the generateConsensus. Sequences kept for further investigations were the ones possessing a mean depth of coverage ≥ 500 and a percentage of gaps ≤ 20% of the entire sequence. For variant calling, variants with a genotype quality (GQ) score ≥ 30, a coverage (FDP) ≥ 500 and a minimum alternate allele frequency of 70% (AF ≥ 70%) were kept for further investigations. Clade and lineages of 479 specimens were defined with the Nextclade v2.29.1 web application (https://clades.nextstrain.org/), the GISAID CoVsurver mutation App (https://www.gisaid.org/epiflu-applications/covsurver-mutations-app/), the pangolin web app developed by the Centre for Genomic Pathogen Surveillance–version v.3.1.11 - (https://pangolin.cog-uk.io/) and the Ultrafast Sample placement on Existing tRee (UShER) web tool by UCSC (https://hgwdev-angie.gi.ucsc.edu/cgi-bin/hgPhyloPlace). SNPs and INDELs frequencies were evaluated in the CovidMiner data portal (https://covid-miner.ifo.gov.it/app/home) and in the GISAID database (https://www.gisaid.org/).

FASTA sequences have been uploaded in the GISAID database. S2 Table enlists Accession IDs and virus names.

Phylogenetic analysis

FASTA sequences were aligned with ClustalW. The Maximum Likelihood (Tamura-Nei method) method was used to generate a phylogenetic tree of the 479 sequences. Alignment and tree were computed with Molecular Evolutionary Genetics Analysis (MEGA) v.11.

Statistics

Nonlinear regression was performed mimicking a dose-response plot. Best-fitting curve was superimposed with least squares fit. All statistical analysis were performed with GraphPad Prism 6.0.

Results

The University Hospital of Udine is the hub that serves the 134 cities that are part of the former province of Udine (Italy) and has tested approximately 262’144 specimens since the third wave of the pandemic in January 2021. We retrospectively analyzed 1508 specimens, tested positive between January 1st, 2021 and April 15th, 2021 to track the occurrence and the potential diffusion of the SARS-CoV-2 variants of concern (VOC) and the variants of interest (VUI), reported by the World Health Organization (WHO) since December 2020. The presence of p.N501Y and p.E484K Spike mutations was assessed by high resolution melting (HRM). We witnessed a fast and exponential growth in the percentage of samples possessing the p.N501Y mutation. The first positive samples were already evidenced in January 2021 (N = 3) with a steady increase in the weeks that followed. Fig 1 panel A-D graphically represents the p.N501Y spread during January and February in our territory. Indeed, 99.1% of p.N501Y positivity was then reached in early April. Table 1 and Fig 1 panel E summarize the percentage of p.N501Y positive samples over the selected analytical time points. Indeed, our data agree with the so called “one-month rule of the B.1.1.7 take-over”, according to which the relative abundance of this lineage goes from 20 to 80% in about four weeks [12, 13].

Fig 1

Expansion of Spike p.N501Y positive samples in Friuli Venezia Giulia.

A-D, p.N501Y spread in the province of Udine. Green dots represent p.N501Y positive samples from January 1st to January 15th (A), from January 16th to January 31st (B), from February 1st to February 15th (C), from February 16th to February 28th (D). E, relative abundance of the p.N501Y positive samples in about 15 weeks. Dots represent the estimated frequency in each fortnight. Dotted lines show 95% confidence interval. Created with Biorender.com.

Table 1

Retrospective distribution of SARS-CoV-2 Spike p.N501Y positive samples in our cohort.

Time interval	Samples analyzed (N)	p.N501Y positive (N)	Percentage (%)
January 1st—January 15th	288	1	0.3
January 16th–January 31st	304	2	0.7
February 1st–February 15th	367	41	11.2
February 16th–February 28th	152	95	62.5
March 1st -March 15th	174	157	90.2
April 1st -April 15th	223	221	99.1

Furthermore, samples were randomly selected and analyzed by next generation sequencing (NGS) to confirm which lineage they belonged to and to obtain the entire genomic sequence. After quality filtering, 479 sequences were retained for further analysis. The mean coverage was 5158 X [95% CI: 4925–5392], with a mean uniformity of sequencing of 94.14% [95% CI: 93.72–94.55]. Indeed, all of the SARS-CoV-2 B.1.1.7 genomes analyzed in our cohort contained all the so-called signature mutations in the Spike glycoprotein, including p.H69-V70del, p.Y144del, p.N501Y, p.A570D, p.P681H, p.T716I, p.S982A, and p.D1118H [3]. Noteworthy, two specimens collected in mid-February turned out to possess the p.E484K but not p.N501Y. After pangolin web app annotation both samples turned out to belong to the B.1.1.318 lineage. Indeed, it was defined as a variant of interest (VUI202102/04) in February 2021 and, since April 2021, 536 cases have been defined worldwide, with a majority in the US, UK, and Germany (https://cov-lineages.org/lineages/lineage_B.1.1.318.html). To get a more insight in the distribution of lineages within our territory, a phylogenetic analysis of the 479 sequences has been performed (Fig 2, panel A). Relative abundance of each lineage is summarized in Fig 2, panel B. Eventually, our 479 sequences have been analyzed with the Nextclade online tool in order to map them within the SARS-CoV-2 global landscape (Fig 2, panel C).

Fig 2

Phylogenetic analysis and clades distribution of positive samples.

A, phylogenetic tree of 479 sequences based on the maximum likelihood method of MEGA v.11. B, distribution of PANGO lineages between January 1st and April 15th in Friuli Venezia Giulia. C, clades distribution according to the Nextclade online tool. Panel A and B share the same color code. Created with Biorender.com.

Subsequently, samples have been analyzed in order to putatively pinpoint whether there was any enrichment in non-synonymous mutations rather than the B.1.1.7-defining ones. 39% of sequences harbored NSP12b p.P218L (Orf1ab p.P4619S), NSP14 p.P451S (Orf1ab p.P6376S), Orf6 p.D61L and Orf8 p.K68* mutations. 20% of viral genomes analyzed presented the NSP4 p.L206F (Orf1ab p.L2969F) in combination with N p.A156S. 14.5% presented the combination of NSP3 p.E405A (Orf1ab p.E1223A) and Orf7a p.V106L. Among these mutations, only the NSP12b p.P218L is already indexed on the CovidMiner data portal with a frequency of 16% among the deposited sequences. Furthermore, the Nucleocapsid p.A156S substitution has been already discussed in the study of Rahman and colleagues, highlighting a mutation-induced shift in protein stability with a decrease in flexibility. Indeed, it has been demonstrated that A156 is among the critical residues that interacts with diverse drug candidates (i.e., Conivaptan, Ergotamine, Venetoclax, Rifapentine), meant to interfere with Nucleocapsid-related functions.

We, then, focused our attention of mutations occurring on the Spike glycoprotein. In the first four time points, we only detected two SNPs that differs from the B.1.1.7-defining ones: the p.D138H substitution (N = 3) and the p.D796H (N = 1). Analyzing the last two time points, in concurrence with the exponential growth in B.1.1.7 lineage frequency, a higher number of Spike mutations have been uncovered. Table 2 and Fig 3 summarize our findings.

Table 2

Novel spike mutation detected in the B.1.1.7 lineage genome background.

Spike mutation	Number of genomes	Occurrence in the cohort	Percentage in CovidMiner
p.L5F	4	1.06%	2.20%
p.V11F	1	0.26%	n.a.
p.L18F	1	0.26%	7.50%
p.T19I	1	0.26%	n.a.
p.P26L	1	0.26%	0.10%
p.A67V	1	0.26%	n.a.
p.I68_H69del	1	0.26%	n.a.
p.V70F	1	0.26%	n.a.
p.D138H	3	0.79%	0.20%
p.D138_Y145del	1	0.26%	n.a.
p.D215Y	1	0.26%	n.a.
p.P330S	1	0.26%	n.a.
p.P384S	4	1.06%	n.a.
p.E484K	2	0.53%	1.50%
p.E583D	3	0.79%	0.30%
p.V608I	1	0.26%	n.a.
p.N658K	1	0.26%	n.a.
p.A694V	1	0.26%	n.a.
p.N751S	1	0.26%	n.a.
p.V772I	1	0.26%	0.50%
p.L922F	1	0.26%	n.a.
p.V1065L	2	0.53%	n.a.
p.P1079S	1	0.26%	n.a.
p.H1101Y	1	0.26%	n.a.
p.V1104L	1	0.26%	n.a.
p.D1153H	1	0.26%	n.a.
p.V1264L	1	0.26%	0.10%

Fig 3

Schematic representation of the Spike glycoprotein and the rare mutations highlighted by NGS.

Cartoon describing the SARS-CoV-2 Spike glycoprotein 3D structure and its interaction with the cellular plasma membrane together with a graphical representation of Spike-related sequencing data. RBD: RNA binding domain; NTD: N-terminal domain; FP: fusion protein; TA: transmembrane anchor; IT: intracellular tail; S1: subunit 1; S2: subunit 2; HR1: heptad repeat region 1; HR2: heptad repeat region 2; the inverted black triangle represents the amino acid bridge. Created with Biorender.com.

It should be noticed that most of these substitutions are rare mutations, highlighted in a single specimen. This prevents us from making any epidemiological or clinical conclusions on the putative consequence of these variants.

Discussion

The emergence of diverse variants of concern in the ongoing SARS-CoV-2 pandemic requires rapid genomic, epidemiological, and clinical characterization to convey public health responses and reorganize hospitalization. We retrospectively investigated the presence of the three previously reported VOC (B.1.1.7, B.1.351 and P1) in a cohort of 1508 specimens, found positive for SARS-CoV-2 infection in a time span of about 4 months in the northeastern area of the Italian peninsula. 479 samples were, then, randomly selected to undergo NGS. In this work, we chose to apply a primary HRM-based screening, followed by the analysis of only a third of samples by NGS. Surely the greatest benefit in using HRM is the possibility to process a large number of samples at a minimum cost and in a very short time, which is not possible using NGS. The latter, in fact, although it gives more informative results in terms of classification of viral sequences and in the discovery of novel mutations and new lineages, is time consuming and requires a huge economic and technological effort that makes it difficult to be applied for the timely tracking of VOC/VUI-infected patients. In our opinion, the combined use of these two techniques is a good compromise that has the ultimate goal of focusing efforts on samples that deviate from the endemic lineages of a certain area in a specific timeframe.

From the beginning of the pandemic, several containment measures were taken from the Italian Government. In particular, during late 2020, Italy was divided into three major risk-zones, calculated by the Italian technical scientific committee taking in account several monitoring objective parameters (i.e., reproduction number (R) and growth rate (r)). Concurrently, in order to minimize risks, the whole nation was considered as a ’high risk zone’; long-ranging transfers and all private gatherings involving more than two persons from different families were banned by law [14]. In January 2021, a gradual mitigation of pandemic-related containment measures, and the consequent opening of the boarders, was authorized.

In this study, we showed that the B.1.1.7 lineage rapidly spread becoming the prevalent lineage from January to March 2021. Indeed, a progressive increase in positive cases of SARS-CoV-2 infections and the concomitant emergence of the Alpha variant were reported in diverse regions of Italy [6, 7, 15, 16]. Lai and colleagues reported that the B.1.1.7 lineage significantly increased from 3.5% in December 2020 to 86.7% in March 2021, replacing the previously circulating variant B.1.177 [7]. Moreover, they assessed that the most important increase in its prevalence was observed from the second week of January to the end of the same month, reaching 73.7% prevalence at the end of February. Our data perfectly fits in this overview.

Thus, it is tempting to speculate that the relaxation of pandemic-related containment measures and the opening of international travels may be implicated in the emergence and subsequent spread of the Alpha variant in our region.

Moreover, our findings are consistent with those gathered worldwide [17] where this lineage established itself as the dominant one within a few months after its detection. Indeed, it has been shown that the p.N501Y mutation is associated to an augmented affinity to the angiotensin-converting enzyme 2 (ACE2) receptor [18] and an increased transmissibility compared to previous circulating SARS-CoV-2 lineages [19]. Notwithstanding, no strong evidences have been made on an increased clinical severity or immune escape capability in patients infected by the B.1.1.7 lineage [20-22]. Indeed, epidemiological data from the former province of Udine highlighted a rapid increase in infection and related hospitalizations after the stabilization of the B.1.1.7 lineage within the territory (Fig 4).

Fig 4

Epidemic curve of confirmed SARS-CoV-2 infected patients in Udine by day.

Blue bars represent the number of samples tested positive for SARS-CoV-2 infection per day between January 1st to April 15th, the period covered by our analysis. The green line represents the number of hospitalized patients due to COVID-19 per day from mid-February. The purple line represents the trend line of daily infections.

Once reached the plateau (i.e., > 90% prevalence), our data showed the onset of several non-canonical mutations on the B.1.1.7-related Spike sequence, a possible hint of the fact that the viral genome is still in continuous evolution. Of particular concern is the B.1.1.7 lineage harboring the p.E484K, since it has been demonstrated that it enhances escape from neutralizing antibody inhibition in vitro, and reduces efficacy of the vaccine [23]. Nonetheless, the presence of the p.E484K mutation in the B.1.1.7 background has been recently reported, named VOC-21FEB-02 in late March 2021. p.E484K is a mutation of concern with regards to antigenic change and receptor binding avidity; it is potentially more concerning when combined with N501Y. It has been first identified in mid-February and it has been postulated to be selected by convalescent and vaccine-derived antisera.

Indeed, mRNA based (Moderna, Pfizer-BioNTech), adenoviral based (Vaxzevria, Johnson&Johnson), and protein-based based (Novavax) vaccines are only able to induce a monoclonal response towards the Asp614Gly bearing SARS-CoV-2 Spike protein, therefore a steady acquisition of mutations that could eventually lead to an enhanced fitness should be carefully monitored.

Conclusions

Despite limitations, we were able to depict the geographic spread and genomic evolution of the SARS-CoV-2 B.1.1.7 lineage in our territory. Our data are a proof of concept that increased genomic surveillance will be crucial for detection of emerging variants of concern, which could escape natural- and vaccine-induced immunity, and for the coordination of appropriate health measures to contain the infection.

Approximate correlation between the major SARS-CoV-2 PANGO lineage, GISAID Nextstrain clade and WHO nomenclature.

(DOCX)

Click here for additional data file.

Accession ID and virus names of the 479 sequences uploaded in the GISAID database.

(DOCX)

Click here for additional data file.

7 Sep 2021

PONE-D-21-25721Local occurrence and fast spread of B.1.1.7 lineage: a glimpse into Friuli Venezia GiuliaPLOS ONE

Dear Dr. Curcio,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Ma conclusion = major revision and The results are of interest as indicated by the reviewerw but some details are lacking. First of all, please made all data underlying the findings in the manuscript fully available. Ie list the sequence accession number and specific list. Explain what is the reference sequence in full and please answer to all the detailled question from reviewers. Please note that specific comment from reviewer 1 is in attachement.

Please submit your revised manuscript by Oct 22 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Pierre Roques, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. You indicated that you had ethical approval for your study. In your Methods section, please ensure you have also stated whether you obtained consent from participants included in the study or whether the research ethics committee or IRB specifically waived the need for their consent.

3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

4. We note that Figure 1 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Overview of the manuscript’s

On real-time screening is the cornerstone strategy to fight against current Covid pandemic, especially variants sequencing. Variants increase transmissibility, virulence. Their association to fatal diseases progression justifies the relationship between variants progression and the hospitalization cases number in this paper. Is the detection of new mutations important, here? Giving more details in the clinical signs of patients (with B.1.1.7 detection), in lineages diversity in this region and at frontiers should be benefices to paper.

Confer attachments to recommendation

Reviewer #2: The paper by Mio et al. describes the emergence and spread of the SARS-CoV-2 alpha variant between January and April 2021 in the Udine region of Italy. The presence of the alpha variant was assessed by HRM as well as NGS, however, in its current form it is in need of a major review, before being suitable for publication.

Comments:

1. Was the typing of all samples only based on the HRM (i.e. presence/absence of these two mutations) or were they all confirmed by NGS?

2. Are there other strains that possess these two mutations but do not belong to the alpha variant?

3. The NGS part is not presented in a clear manner. How many samples were analysed? What were the criteria for choosing these samples?

4. I would suggest to add a phylogenetic analysis for the sequences obtained including sequences of surrounding regions during the same time period. Also, the accession numbers are missing.

5. Is clinical data available for this time period? It would be interesting to evaluate the impact of the spread of the alpha variant with regard to hospitalisations, severity of disease course and mortality.

6. The discussion needs expansion. What happened in this time period in other parts of Italy (or Europe) with regard to the alpha variant?

7. In many parts I had to struggle with the language; I am not sure what “private” mutations refer to or how samples were “harvested”. I strongly suggest that the manuscript is proof-read by a native English speaker before resubmission

8. Table 1 should be moved to supplementary information; the WHO classification should be indicated in a separate column instead of in brackets in the Nextstrain clade column

Reviewer #3: The manuscript from Mio et al. retrospectively investigated the local occurrence and spread of B.1.1.7 in a region in Northeastern Italy. The information provided in this manuscript can be useful for understanding the local pandemic development. However, some detailed information should be added. I would like to suggest revisions regarding the topics below:

1. About data availability:

The authors describe “sequences are available from the GISAID database”, without providing detailed information. For the 1508 sequences, the authors should provide GISAID Accession ID, so anyone who read the paper could directly check the sequences.

2. In Table 1, the authors summarised mutation information for several variants. The authors did not describe which reference genome they refer to when discussing mutations. The “reference genome” information should be provided, because the mutations can be different if a different reference genome is used in the analysis.

3. In Table 2, information about the time interval “March 16th - March 31th” is missing. Any specific reason for that? Otherwise, the authors should provide relevant information for this time period as well.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Jan Richter

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: PONE-D-21-25721-Recom.pdf

Click here for additional data file.

22 Oct 2021

Point-by-point answers to the reviewer’ requests:

Reviewer #1:

Comments to the Author:

Overview of the manuscript’s : On real-time screening is the cornerstone strategy to fight against current Covid pandemic, especially variants sequencing. Variants increase transmissibility, virulence. Their association to fatal diseases progression justifies the relationship between variants progression and the hospitalization cases number in this paper. Is the detection of new mutations important, here? Giving more details in the clinical signs of patients (with B.1.1.7 detection), in lineages diversity in this region and at frontiers should be benefices to paper.

Summary of the research

The researcher group takes the opportunity to combine High Resolution Melting (HRM) approach and Next Generation Sequencing analysis (NGS) to screen the first four months of year and bring an overview to virus genomic evolution from January 1st 2021, until April 15th 2021 in this particular north-eastern region of Italy. The conclusion is an increasing of variant for this period as well as cluster sub-lineages with interest concern B.1.1.318 lineage.

Keys list:

Strengths

Sequenced samples number

Screening methods combination (HRM approach and sequencing)

Ethical guidelines respect

Spatio-temporal information of reported cases

Weaknesses:

Short duration of study (4 months)

Unuseful work in new mutation detection

No clinical signs data and demographic information of cases

No advices and perspectives to push interest of this paper

Recommended course of action (major):

1. Introduction.

People speak about 19 A 20 A clades (line 40-41) without referring to Nextstrain nomenclature and its references (add website link and version).

The website and the relative version have been added to the revised version of the manuscript (page 3, line 45).

2. In addition, it needs to recall that Pangolin nomenclature system is not a way to “solve a problem” to GISAID or Nextstrain assignement supports. Pangolin is a complementary approach to other, and in especially to focus on the local dynamic and through lineages diversity instead of clades. Conjugating nomenclature systems are so interesting method. Don't forgot also WHOs' (Alpha, Beta, etc..). It needs to be mentioned in the introduction too.

The paragraph discussing the nomenclature system has been rewritten, including the WHO-related classification (page 3, lines 47-56).

3. Epidemiological context of pandemic in Italy and in this specific region prior of the study should be mentioned at this time point. This is to better understand the possible consequence in variants emergence and paper' interest.

We thank the reviewer for the suggestion. Retrospective studies highlighted that in Italy the main circulating lineage were B.1 and B.1.177 together with their sub-lineages, representing about 70% of cases in late 2020. In our region, the prevalent lineages were the B.1.177 and B.1.258 ones, according to the sequences related to November-December 2020 uploaded in the GISAID database. This information has been added to the text (page 7, lines 61-63) together with appropriate references.

4. Material and methods

Precise the process of samples selection for 4 months: random n samples by months, no rule and only available samples?

Samples were randomly selected from all the SARS-CoV-2 swabs tested positive in the timeframe analysed in this study. This sentence has been added in the new version of the revised manuscript (page 7, line 77-84).

5. Add demographical and clinical information related to sequencing samples

We thank the reviewer for the suggestions to improve our manuscript. Unfortunately, we are not in possession of the cohort-related clinical data.

6. Give mor info in High resolution melting (HRM) technic and its benefices: melting temperature analysis...

Thermal shifts have been added to the revised version of the manuscript, to better comprehend this method (page 8, lines 97-98).

7. Notice date of assignement by using Nextstrain and Pangolin (line 101 103) due to update in Continuous

Databases and tools versions have been included in the material and methods section (page 9, lines 121-129).

8. Results

Lines 108- 115 need to be moved to in material ad methods

We thank the reviewer for the suggestions to improve our manuscript. The aforementioned sentences have been edited and partially added to the material and methods section (page 7, lines 77-84).

9. Data shows only lineages reports (Pangolin assignement). Its of Nextstrain assignement miss and added concerning 1508 seq (i.e.: is it clade 20Ia and for all?)

Since PANGO nomenclature turned out to be the most widely used (https://doi.org/10.1038/s41576-021-00408-x), before the WHO reclassification, which is indeed posterior to the timeframe described in this paper, we preferred to use one nomenclature only. Notwithstanding, PANGO lineages and Nextsrain clades have been summarized in Figure 2.

10. Figure 2: most of lineage are B.1.1.7 lineages (major %). but what about the % remaining?

Data related to lineage distribution are now summarized in Figure 2.

11. Figure 1: It shows delay between infected patients (blue line) and hospitalizations number (green line). In addition, it should be possible to link the increasing in variants incidence to specific clinical signs changes and/or demographical population targeted (ages, sex etc..) for 4 months at hospital (i.e: table with progression index, age, sex, etc...). It provides opportunity to perform additionnal multivariable statistical analysis in correlation between to these events.

We thank the reviewer for the suggestions to improve our manuscript. Unfortunately, we are not in possession of the cohort-related clinical data.

12. In contrast, work in new mutations detection has not to be place on main document. It makes confusing analysis without arguing in interested to support main message. This part may be rewrited by only speaking in known mutations concern to variant leading to review figure 1 C/D and Table 2 to suppl.

We thank the reviewer for the suggestions to improve our manuscript. Since the continuous monitoring in the establishment of novel mutation is the key to overcome the occurrence of future variants of concern, we believe that tracking novel and rare mutation is crucial in the pandemic outbreak.

13. Discussion

Arguing in benefices to combine HRM + sequencing approaches, or oppose to it, as a timeconsuming as well as too restrictive (no new variants discovery) approach compared to directly perform sequencing of all positive samples. NGS allows to open the opportunity to detect other variants here, already described in worldwide and to frontiers as well as to propose under monitored variants as concern. Moreover, it is surprising to only detect 2 lineages B.1.1.7 and B.1.1.318. In general, SARS-Cov-2 sequencing in other countries shows diversity in lineages more and even in clades. This poorness is it justify or not (among >1500 seq.)? No detection of parental lineages (i.e: B.1) ?

We implemented the Discussion section arguing pro and cons of the approach used in this manuscript (Pages 15-16, lines 238-247). Moreover, a better description on lineage heterogeneity in sequenced samples together with a representative figure have been added to the text.

14. Speak in possible causes of variant introduction at the beginning of year by providing information to reported cases and/or to frontiers regions (business exchanges, traveler, unlock-down event, vacancy…).

Since we do not possess epidemiological data related to our cohort, we prefer not to speculate on the possible way in which the alpha variant has established itself in our territory.

15. In line 50, people mention “Viral genome sequencing coupled to epidemiological surveillance is mandatory”, it is a pity that the sequencing is not associated to epidemiological during the study period to discuss on it here.

Unfortunately, we do not possess these data.

16. Conclusion

No advice and/or perspectives to use sequencing at hospital (to management) as well at frontiers to target new variant introductions in this strategic part of the Italian region.

Considerations of using an NGS-based screening protocol were included in the discussion section of our manuscript. NGS, indeed gives more informative results in terms of classification of viral sequences and in the discovery of novel mutations and new lineages. Nonetheless, it is time consuming (72 hours are needed to obtain lineage information) and it requires a huge economic and technological effort that makes it difficult to be applied for the timely tracking of VOC/VUI-infected patients.

17. Graphics

Table 1: this relationship between GISAIS/Nextstrain and Pangolin nomenclature systems is well described in Alma et al, (https://doi.org/10.2807/1560-7917.ES.2020.25.32.2001410). Paper could be noticed to remove table 1. Or table1 should be redesigned (Column 1 GISAID, Column 2 Nextstrain, Colum 3 Pangolin) and put on suppl. In addition, table 1 needs more legend details (choice of some variants and not others, mutations code (p.?) and also mentioned WHO nomenclature (alpha, beta etc..).

The aforementioned paper has been added to the manuscript text and Table 1 has been moved to Supplementary Data.

18. Figure 1 1A: 4 maps, one for each month, allow to localize positive cases in space (in green), identify some of them associated to variant (in black) and possibly show cluster emergence in time (i.e. radiation or centralization). 1B: it is related to table 3 and better to put together one above other. 1C and 1D are structural and mutations concern: to put apart in a new figure, especially to suppl. as already recommended.

We thank the reviewer for the suggestions to improve our manuscript. Figures have been edited to better describe our data.

Recommended course of action (minor):

1. Introduction it may be important to justify the neutral genetic drift (line 33) and enhance of viral fitness affect in mutations constellations (line 53) with SARS-Cov-2 references.

Appropriate references have been added to the main text.

2. Methods

% of targeted coverage, number of sequencing with >10% missing genome (no success).

Data relative to NGS-based data analysis have been added to the main text.

3. Results

Bring information in patient treatments and management results to variants emergence at hospital. It might affect the quality of diagnostic and/or sequencing success?!

Unfortunately, we do not possess these data.

4. Discussion

The emergence of variant is it associate to guidelines changes in local regions such as lockdown, diagnostic or vaccine recommendation?

From the beginning of the pandemic, several containment measures were taken from the Italian Government. In particular, during late 2020, Italy was divided into three major risk-zones, calculated by the Italian technical scientific committee taking in account several monitoring objective parameters (i.e., reproduction number (R) and growth rate (r)). Concurrently, in order to minimize risks, the whole nation was considered as a 'high risk zone'; long-ranging transfers and all private gatherings involving more than two persons from different families were banned by law. In January 2021, a gradual mitigation of pandemic-related containment measures, and the consequent opening of the boarders, was authorized. Indeed, a progressive increase in positive cases of SARS-CoV-2 infections and the concomitant emergence of the Alpha variant were reported. Thus, it is tempting to speculate that the relaxation of pandemic-related containment measures and the opening of international travels may be implicated in the emergence and subsequent spread of the Alpha variant in our region. This information has been added to the text (page 16, lines 250-266) together with appropriate references.

5. Remove 188-195 lines as losing the lector to main goal

We thank the reviewer for the suggestion. Notwithstanding, we believe that the continuous monitoring and recording of new rare mutations is mandatory to comprehensively investigate and keep track of virus evolution and could shed light on possible causes of reduced vaccine efficacy. Therefore, we prefer to keep this section in our manuscript.

6. Figure Improve the resolution quality of figures

As described in the journal guidelines, higher resolution images will be introduced if the manuscript is accepted for publication.

Reviewer #2

Comments to the Author:

The paper by Mio et al. describes the emergence and spread of the SARS-CoV-2 alpha variant between January and April 2021 in the Udine region of Italy. The presence of the alpha variant was assessed by HRM as well as NGS, however, in its current form it is in need of a major review, before being suitable for publication.

Comments:

1. Was the typing of all samples only based on the HRM (i.e. presence/absence of these two mutations) or were they all confirmed by NGS?

1508 samples were screened by HRM and then randomly selected for sequencing. After quality-based filtering, 479 sequences were retained for further analysis (Page 8, line 101). A more detailed workflow has been added in the new version of the manuscript.

2. Are there other strains that possess these two mutations but do not belong to the alpha variant?

The analysis of the sequencing data did not reveal other strains possessing both p.E484K and p.N501Y other than the alpha lineage. A summary of the non-alpha lineages highlighted in our study has been included in the new version of the manuscript (Figure 2).

3. The NGS part is not presented in a clear manner. How many samples were analysed? What were the criteria for choosing these samples?

We thank the reviewer for the suggestions to improve our manuscript. A better description of the NGS-based workflow has been added in the material and methods session. Samples underwent HRM screening, were randomly selected for NGS. After filtering criteria based on coverage (FDP>500), percentage of gaps (N< 20% of the entire sequence), 479 sequences were retained for further analyses.

4. I would suggest to add a phylogenetic analysis for the sequences obtained including sequences of surrounding regions during the same time period. Also, the accession numbers are missing.

A phylogenetic analysis of the 479 sequences included in our manuscript has been added (Figure 2). The list of the GISAID Accession ID is now available as Supplementary Table 2.

5. Is clinical data available for this time period? It would be interesting to evaluate the impact of the spread of the alpha variant with regard to hospitalisations, severity of disease course and mortality.

We thank the reviewer for the suggestions to improve our manuscript. Unfortunately, do not possess the cohort-related clinical data.

6. The discussion needs expansion. What happened in this time period in other parts of Italy (or Europe) with regard to the alpha variant?

We thank the reviewer for the suggestion. Retrospective studies highlighted that in late 2020 in Italy the main circulating lineage were B.1 and B.1.177 together with their sub-lineages, representing about 70% of cases. From December 2020, a progressive increase in positive cases of Alpha-related infections were reported in different part of Italy, with a trend overlapping the one presented in our manuscript. Indeed, Lai and colleagues reported that the B.1.1.7 lineage significantly increased from 3.5% in December 2020 to 86.7% in March 2021 in diverse Italian regions [doi: 10.1186/s12985-021-01638-5]. Moreover, they assessed that the most important increase in its prevalence was observed from the second week of January to the end of the same month, reaching 73.7% prevalence at the end of February. Our data perfectly fits in this overview. This information has been added to the text (page 16, lines 250-266) together with appropriate references.

7. In many parts I had to struggle with the language; I am not sure what “private” mutations refer to or how samples were “harvested”. I strongly suggest that the manuscript is proof-read by a native English speaker before resubmission.

In genetics, “private mutations” are variations found usually only in a single family or a small population. Notwithstanding, the description of rare SNP identified in our cohort has been rewritten, to avoid any possible confusion. Moreover, the new version of the manuscript has been entirely revised for correct use of language and for typos.

8. Table 1 should be moved to supplementary information; the WHO classification should be indicated in a separate column instead of in brackets in the Nextstrain clade column.

Table 1 has been modified and moved to supplementary data.

Reviewer #3

Comments to the Author:

The manuscript from Mio et al. retrospectively investigated the local occurrence and spread of B.1.1.7 in a region in Northeastern Italy. The information provided in this manuscript can be useful for understanding the local pandemic development. However, some detailed information should be added. I would like to suggest revisions regarding the topics below:

1. About data availability: The authors describe “sequences are available from the GISAID database”, without providing detailed information. For the 1508 sequences, the authors should provide GISAID Accession ID, so anyone who read the paper could directly check the sequences.

We thank the reviewer for the suggestion. 1508 samples were screened by HRM and then randomly selected for sequencing. A more detailed workflow has been added in the new version of the manuscript. About 479 sequences were uploaded in the GISAID database. The list of the GISAID Accession ID is now available as Supplementary Table 2.

2. In Table 1, the authors summarised mutation information for several variants. The authors did not describe which reference genome they refer to when discussing mutations. The “reference genome” information should be provided, because the mutations can be different if a different reference genome is used in the analysis.

We thank the reviewer for the suggestion. The reference genome used in this manuscript is the NC_045512.2 assembly. This information has been added in the material and methods session (Page 8, line 105).

3. In Table 2, information about the time interval “March 16th - March 31th” is missing. Any specific reason for that? Otherwise, the authors should provide relevant information for this time period as well.

Samples related to the aforementioned time point have not been analyzed and therefore this interval is missing in Table 2.

Submitted filename: Response to reviewers_Plosone_21-10-2021.docx

Click here for additional data file.

25 Nov 2021

Local occurrence and fast spread of B.1.1.7 lineage: a glimpse into Friuli Venezia Giulia

PONE-D-21-25721R1

Dear Dr. Curcio,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Pierre Roques, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: During the review all points raised previously have been addressed in a satisfactory manner. The manuscript is now deemed acceptable for publication.

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Jan Richter

Reviewer #3: No

3 Dec 2021

PONE-D-21-25721R1

Local occurrence and fast spread of B.1.1.7 lineage: a glimpse into Friuli Venezia Giulia

Dear Dr. Curcio:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Pierre Roques

Academic Editor

PLOS ONE