SARS-CoV-2 lineage B.6 was the major contributor to early pandemic transmission in Malaysia.

Malaysia had 10,219 confirmed cases of COVID-19 as of September 20, 2020. About 33% were associated with a Tablighi Jamaat religious mass gathering held in Kuala Lumpur between February 27 and March 3, 2020, which drove community transmission during Malaysia's second wave. We analysed genome sequences of SARS-CoV-2 from Malaysia to better understand the molecular epidemiology and spread. We obtained 58 SARS-CoV-2 whole genome sequences from patients in Kuala Lumpur and performed phylogenetic analyses on these and a further 57 Malaysian sequences available in the GISAID database. Nine different SARS-CoV-2 lineages (A, B, B.1, B.1.1, B.1.1.1, B.1.36, B.2, B.3 and B.6) were detected in Malaysia. The B.6 lineage was first reported a week after the Tablighi mass gathering and became predominant (65.2%) despite being relatively rare (1.4%) globally. Direct epidemiological links between lineage B.6 viruses and the mass gathering were identified. Increases in reported total cases, Tablighi-associated cases, and community-acquired B.6 lineage strains were temporally linked. Non-B.6 lineages were mainly travel-associated and showed limited onward transmission. There were also temporally correlated increases in B.6 sequences in other Southeast Asian countries, India and Australia, linked to participants returning from this event. Over 95% of global B.6 sequences originated from Asia Pacific. We also report a nsp3-C6310A substitution found in 47.3% of global B.6 sequences which was associated with reduced sensitivity using a commercial diagnostic real-time PCR assay. Lineage B.6 became the predominant cause of community transmission in Malaysia after likely introduction during a religious mass gathering. This event also contributed to spikes of lineage B.6 in other countries in the Asia-Pacific. Mass gatherings can be significant causes of local and global spread of COVID-19. Shared genomic surveillance can be used to identify SARS-CoV-2 transmission chains to aid prevention and control, and to monitor diagnostic molecular assays. Clinical Trial Registration: COVID-19 paper.

Introduction

Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused more than 33 million infections and 1 million deaths globally [1]. In Malaysia, there have been 10,219 confirmed cases with 130 deaths reported as of September 20, 2020 [2], out of a total population of 32.7 million, giving a cumulative incidence of 31 per 100,000. COVID-19 was first identified in Malaysia on 25 January 2020 with early sporadic cases mainly associated with travel from China and Singapore. The second wave of infection began in early March 2020, and was associated with attendance at a religious mass gathering held by the Tablighi Jamaat group in Kuala Lumpur between 27 February to 3 March. This gathering was attended by 16,000 people, including about 1,500 from overseas, including Australia, Canada, Nigeria, and India, and neighbouring Southeast Asian countries Singapore, Indonesia, Thailand, Cambodia, Vietnam, Brunei and Philippines [3]. As the number of confirmed cases rapidly increased, Malaysia had the highest number of confirmed cases in Southeast Asia in March and early April. A movement control order, or lockdown, restricted movement except for necessity, work, and health circumstances from 18 March. Through rigorous public health measures, case numbers began to fall, allowing phased lifting of restrictions after 3 months.

SARS-CoV-2 is an RNA virus from the family of Coronaviridae and genus Betacoronavirus. There are currently more than 100,000 publicly available complete or near-complete genome sequences of SARS-CoV-2 with over 100 lineages identified. As the virus is rapidly evolving, it is important to understand the genomic epidemiology of SARS-CoV-2 to track virus evolution and local and global spread.

Here we report the whole genome sequences of SARS-CoV-2 from 58 patients from University Malaysia Medical Centre (UMMC), a designated COVID-19 hospital in Kuala Lumpur. Our objective was to study the genetic diversity and epidemiology of SARS-CoV-2 in Malaysia, relative to the regional and worldwide virus lineages. In particular, we were interested in determining the possible role of the Tablighi Jamaat gathering in national and regional spread. Genomic surveillance improves monitoring and understanding of current SARS-CoV-2 spread within a country and can help inform public health measures.

Methods

Ethics statement

This study was approved by the University Malaya Medical Centre ethics committee (no. 2020730–8928). Our institution does not require informed consent for retrospective studies of archived and anonymised samples.

Clinical sample collection

Patients admitted to UMMC with suspected COVID-19 were diagnosed by real-time PCR detection of SARS-CoV-2 in nasopharyngeal and oropharyngeal swabs, using a WHO-recommended Berlin Charité protocol [4] and commercial assays. For each patient, the earliest available sample during illness with the highest viral load was selected for direct sequencing. Cases were diagnosed between February and April 2020.

Whole genome sequencing analysis

Viral RNA was extracted from 58 positive clinical samples using QIAamp Viral RNA Mini Kit (Qiagen, Germany) and amplified according to the ARTIC-nCoV-2019 protocol [5]. Briefly, cDNA was synthesized using SuperScript IV First-Strand Synthesis System (Invitrogen, USA) with random hexamers. Multiplex PCR was performed with Q5 High-Fidelity DNA Polymerase (NEB, USA) using two pools of 109 nCoV-2019/V3 primer sets to generate overlapping 400 nucleotide amplicons. PCR products were pooled into one tube and purified using AMPure XP beads (Beckman Coulter, USA). The iTrue library method was used for library preparation [6]. Libraries were sequenced using iSeq 100 reagent kit (Illumina, USA) on the iSeq 100 system (Illumina, USA), with output of 1 × 300bp reads.

Bioinformatic analysis

The sequenced reads were analysed using Geneious Prime 2020 (Biomatters, New Zealand). Reads were trimmed for quality using default parameters and mapped to reference strain Wuhan-Hu-1 (GenBank accession number MN908947). Leftover gaps were sequenced separately by conventional Sanger sequencing using the nCoV-2019/V3 primers set and consensus sequences were generated. Multiple sequence alignment was performed using MAFFT with default parameters [7]. Phylogenetic analysis was conducted with RAxML 8.2.11 implemented in Geneious with default parameters (generalized time-reversal (GTR) + gamma substitution model and bootstrapped 1000 times). The analysed Malaysian sequences comprised the 58 samples from this study and 57 other Malaysian genome sequences available at GISAID (www.gisaid.org) as of September 20, 2020 [8], which include 4 previously published by our centre [9]. Therefore, 62 of the Malaysian sequences were from our centre. Variant analysis was performed among all Malaysian sequences using Geneious. Unique nucleotide mutations were further confirmed using the CoV-GLUE online tool (http://cov-glue.cvr.gla.ac.uk) [10]. Lineage groups were classified according to the Pangolin COVID-19 Lineage Assigner online tool (www.pangolin.cog-uk.io) [11].

SARS-CoV-2 lineage B.6 complete genome sequences up to September 20, 2020 were retrieved from GISAID. A total of 1,497 B.6 sequences, including 75 Malaysian B.6 sequences (43 from our study) were available in the GISAID database. Of these, 1,122 high quality B.6 sequences with > 29,000 bp, < 1% Ns and < 0.05% unique amino acid mutations were aligned using MAFFT with default parameters in Geneious. The alignment was then subjected to maximum-likelihood (ML) phylogenetic analyses using IQTREE v1.6.12 [12], using the GTR+F+I+G4 nucleotide substitution model and assessing branch support by the Shimodaira-Hasegawa-like approximate likelihood ratio test with 1,000 replicates. The ML tree was visualized using FigTree v1.4 [13].

Results

Fifty-eight whole genome sequences with >99% reads mapped to the reference genome were generated, with average coverage depth of 944× (range, 74× to 7119×; S1 Table). The consensus sequences have been deposited in the GISAID database (accession numbers EPI_ISL_501176 to EPI_ISL_501228, and EPI_ISL_506996 to EPI_ISL_50700). All other Malaysian whole genome sequences (57 sequences) available in GISAID were added to the analysis, making a total of 115 Malaysian sequences.

Nine lineage groups were identified in the 115 Malaysian sequences: A, B, B.1, B.1.1, B.1.1.1, B.1.36, B.2, B.3 and B.6 (Fig 1). The most identified lineage group was B.6 (n = 75, 65.2%), followed by B (n = 21, 18.3%), B.1.1 (n = 5, 4.4%), B.1 (n = 4, 3.5%), A (n = 3, 2.6%), B.1.1.1 (n = 2, 1.7%), B.2 (n = 2, 1.7%), B.1.36 (n = 2, 1.7%) and B.3 (n = 1, 0.9%).

Fig 1

Phylogenetic tree of 115 SARS-CoV-2 whole genome sequences from Malaysia.

Sequences from our study are shown in blue and the 7 sequences with known links to the Tablighi Jamaat gathering are denoted with red circles. The tree is rooted on the branch separating lineages A and B. The key single nucleotide variants in lineage B.6 are highlighted.

Although lineage B.6 (which also belongs to GISAID clade O and Nextstrain clade 19A) was the most common in Malaysia, only 1,497 B.6 sequences had been reported globally out of over 105,568 sequences available as of September 20, 2020, a rate of 1.4%. Even after considering 17 sequences generated from the current study which were part of a healthcare-associated cluster, B.6 remained the most frequently detected lineage in Malaysia. Of the 62 cases from our centre, comprising 58 from this study and 4 from an earlier study [9], 7 had direct links to the Tablighi Jamaat gathering– 4 had attended and 3 were contacts of attendees. All 7 had B.6 lineage viruses, thus establishing a link between the Tablighi Jamaat and the B.6 lineage.

The Tablighi Jamaat lasted from February 27 to March 3, 2020, and the first reported B.6 sequences globally were from Malaysia, Philippines and Taiwan on March 4 (Fig 2A). Within a week of the Tablighi Jamaat ending, there was a large increase in reported COVID-19 cases in Malaysia, with the vast majority being associated with this gathering. This was accompanied by a spike of lineage B.6 in Malaysia and other countries (Fig 2A and 2B). From March 3 onwards, lineage B.6 sequences comprised 46/61 (75.4%) of sequences from our centre and 29/39 (74.4%) of sequences submitted by other Malaysian centres, indicating that this finding was not specific to our centre. This increase in cases led to the imposition of a nationwide movement control order on March 18.

Fig 2

(A) Epidemic curve of COVID-19 in Malaysia and time course of reported total cases, Tablighi-associated cases, and lineages based on 115 Malaysian sequences, from the first reported case on 25 January to 20 September 2020. The Tablighi Jamaat gathering in Kuala Lumpur is labelled as “mass gathering, KL”. The different phases of lockdown are shown, comprising the movement control order (MCO), conditional movement control order (CMCO) and recovery movement control order (RMCO). (B) Time course of global reported lineage B.6 SARS-CoV-2 sequences available in the GISAID database (as of 20 September 2020). The timescale of the horizontal axis is the same as (A). Countries with fewer than 5 lineage B.6 sequences are included in “others”, comprising Brazil (1), China (3), France (1), Gambia (1), Guam (3), Hong Kong (1), Iceland (1), Jamaica (1), Japan (4), Kenya (1), Myanmar (1), New Zealand (3), Oman (4), Portugal (2), Saudi Arabia (2), Senegal (1), Sierra Leone (1), South Africa (4), South Korea (2), Slovenia (1) and Turkey (1). (C) The percentage rates of lineage B.6 sequences over total available complete sequences (in brackets) from each country available at GISAID as of 20 September 2020. Asia Pacific countries are colored grey.

Of the 1,497 globally reported B.6 sequences, 1,422 (95%) originated from Asia Pacific countries. Singapore had the highest number of sequences (664), followed by India (532), Australia (81), Malaysia (75), USA (25), Philippines (16), Timor-Leste (14), Canada (13), United Kingdom (10), Thailand (9), Taiwan (7), Brunei (5) and Israel (5). In several Southeast Asian countries (Malaysia, Singapore, Brunei, Timor-Leste, Philippines and Myanmar), lineage B.6 became predominant and accounted for 50–100% of total sequences in GISAID (Fig 2C). Of the 7 Malaysian B.6 cases with Tablighi links, sequence 188407 and 3998 clustered with sequences from Singapore, India and Australia; sequences 2063 and 5096 clustered with sequences from Singapore; sequence 2875 grouped with sequences from Singapore, India and USA; sequence 2297 linked with Singapore, India and New Zealand; and sequence 1399 clustered with those from India, USA, Oman, Australia, Taiwan, Canada, Guam and Sierra Leone (Fig 3). The appearance and surge of overall COVID-19 cases, Tablighi-associated cases, and B.6 lineage cases in Malaysia were thus temporally linked, as were B.6 lineage cases in other Asia Pacific countries.

Fig 3

Phylogenetic tree of 1,122 high-quality B.6 lineage SARS-CoV-2 sequences with > 29,000 bp, < 1% Ns and < 0.05% unique amino acid mutations available from the GISAID database as of 20 September 2020.

Sequences linked to the Tablighi Jamaat gathering are labelled in blue and denoted with red circles.

Several distinct mutations were observed among the Malaysian B.6 sequences. The 17 sequences in the healthcare-associated cluster had a non-synonymous mutation C25549T (P53F) in ORF3a (Fig 1). A secondary cluster within this healthcare-associated cluster contained six sequences with an additional mutation C21627T (T22I) in the spike protein. Eight Malaysian sequences, including 6 from this study, had the additional substitution C6310A, resulting in the amino acid change S1197R in nsp3. This C6310A mutation led to reduced sensitivity by 1,000–10,000 viral copies (> 10 cycles) with one of the commercial real-time PCR assays used in our laboratory when compared to the well-established Charité assay [4] and updated primers/probes issued by the assay manufacturer in response to this likely sequence mismatch (S2 Table). Some mutations were unique to Malaysian B.6 genomes, such as C2508A, A3223G, C9438T, C9803T, C13329T, C20823T, C26607T, A29086T and C29218T; others, including C6310A, T7621C and C19524T, were also observed in B.6 sequences from other countries including Singapore, Australia and India (Fig 3).

The mutation D614G in the spike protein, which may increase infectivity of SARS-CoV-2 [14] and has become prevalent in many countries, was only observed in 13 (11.3%) Malaysian sequences from lineages B.1, B.1.1, B.1.1.1 and B.1.36 (S3 Table), but was not present in lineage B.6. None of the 41 rare receptor binding variants described in GISAID–including the two most common, S477N and N439K –were observed in the Malaysian sequences.

Discussion

In Malaysia, the Tablighi Jamaat mass gathering became the largest COVID-19 cluster, resulting in the screening of over 42,000 individuals, with 3,375 confirmed cases (33% of national cases) and 34 deaths, of which 29 (85.3%) were aged 50 and above. Multiple generations and at least 17 sub-clusters linked to this gathering were detected [14]. Of the cases, 825 (24.4%) were foreign nationals from 28 countries, including Southeast Asian countries, Australia, India, Pakistan and Bangladesh [15]. Religious gatherings were recognized early in the pandemic as a significant factor in the spread of COVID-19 [16], with large clusters associated with events in India, Iran and South Korea [17-20].

We established a direct epidemiological link between the Tablighi Jamaat event and lineage B.6 viruses for 7 cases. The first reported B.6 viruses date from a week after the Tablighi Jamaat event started. Thus, the appearance of the B.6 lineage, and subsequent surge of overall COVID-19 cases, Tablighi-associated cases, and B.6 lineage cases in Malaysia were temporally linked, as were B.6 lineage cases in other Asia Pacific countries. The diversity of the 7 Tablighi-linked Malaysian B.6 sequences (Fig 3) suggests there may have been multiple B.6 strains involved in the initial exposure, which is compatible with the large number of attendees from many different countries who could have been the sources. The participants spent several days in large indoor congregations with shared eating and sleeping arrangements [3], which would have led to extensive transmissions. After the event, non-Kuala Lumpur residents (Malaysian and foreign) would have returned to their areas of residence.

Attendees at the Kuala Lumpur event also apparently acted as sources of further extensive transmission occurring at two subsequent Tablighi Jamaat gathering in Pakistan and India later in March [21]. This may explain a later rise in B.6 in India in late March (Fig 2B). The first known COVID-19 case in Brunei returned from the Kuala Lumpur mass gathering and 52.6% of subsequently reported Brunei cases were associated with this event as of early April [22]. Southern Thailand also experienced an outbreak of COVID-19 after Thai participants returning from this event had a reported 29.3% attack rate [23]. Other Southeast Asian countries including Cambodia, Indonesia, Singapore, Philippines and Vietnam also reported imported cases associated with this gathering [24-28]. A caveat is that the number of whole genome sequences reported from developing countries in Asia is relatively low and likely underreports the true incidence of B.6. However, it is notable that this lineage is a very minor contributor in developed countries with greater sequencing efforts (Fig 3C). Detailed, publicly available epidemiological data are also not available for most of the B.6 sequences outside our centre, and would be very useful to further support the involvement of Tablighi Jamaat participants.

Whole genome sequencing of SARS-CoV-2 in our centre not only allowed important insights into local and regional epidemiology, but was also used to track networks within a healthcare-associated cluster, which will be reported elsewhere. Furthermore, public sharing of sequences contributed to identification of the C6310A (nsp3-S1197R) mutation affecting the sensitivity of a commercial diagnostic real-time PCR assay, leading to updated primers/probes. This mutation is present in only 0.7% of global sequences, but as many as 708 (47.3%) of 1,497 available lineage B.6 sequences, and within our centre we estimate that the reduced sensitivity of the original assay impacted 10% of positive samples. With many diagnostic kits now on the market and the continued evolution of the virus, this shows the importance of choosing reputable manufacturers who are open about the primer and probes used [29] and diligently monitor new circulating sequences for potential primer/probe mismatches [30]. Assays targeting more than one gene are also preferable [29].

There were 40 (34.8%) non-B.6 lineage sequences reported from Malaysia, including 15 from our study. At least 25 (62.5%) reported recent international travel (including 3 of the earliest cases from lineage A, imported from China), compared to just 2/46 (4.3%) of our centre’s B.6 cases. Our sequenced cases with lineages B.1.1, B.2 and B.3 had travelled from Europe and a single B.1 case had returned from a cruise to the Americas. The B.1 and B.1.1 lineages are the most common in Americas and Europe [31]. The small numbers of other non-B.6 lineages indicate multiple introductions from overseas that failed to establish significant transmission in Malaysia, as the movement control order from 18 March closed the country’s borders and mandated quarantine for all incoming travellers. However, by this point the B.6 lineage had established community transmission, reflected by the local acquisition of disease in most of our B.6 cases without direct or discernible links to the Tablighi Jamaat event.

In summary, initial COVID-19 spread and the predominance of lineage B.6 were temporally and epidemiologically associated with the Tablighi Jamaat religious gathering, and led to established community spread during the second wave in Malaysia. Attendees at this event likely spread strains of the B.6 lineage to other countries in the region, including Southeast Asian countries, India and Australia. Further sequence and epidemiological data are needed from Malaysia and other countries in the region to understand the full extent of the spread of B.6. The use of genomic data is important to rapidly identify possible transmission chains and provide a framework for the response to COVID-19.

List of 115 SARS-CoV-2 genomes derived from Malaysian samples and available in GISAID which were used in this study.

The first 58 sequences were generated in this study. Sequences 77–80 were published by our centre [9].

(DOCX)

Click here for additional data file.

Comparison of threshold cycle (Ct) values obtained using the Berlin Charité RT-qPCR assay (E gene) [4] and commercial RT-qPCR assay (nsp3 gene; original and updated primers/probes) using 16 patient samples from this or a previous study [9] categorised by presence/absence of the nsp3-C6310A substitution.

A difference in viral RNA of one log or 10× in the initial template concentration is equivalent to a Ct difference of 3.3.

(DOCX)

Click here for additional data file.

Nucleotide and amino acid changes in different SARS-CoV-2 lineages in the 115 Malaysian sequences compared to the reference strain Wuhan-Hu-1 (MN908947).

Amino acids are denoted in parentheses and unique mutations found in each lineage are bold and highlighted in grey. The sequence names are colored according to their lineage: Lineage A in red color, lineage B in black color, lineage B.1 in purple color, lineage B.1.1 in pink color, B.1.1.1 in golden color, B.1.36 in brown color, B.2 in orange color, B.3 in green color and B.6 in blue color.

(XLSX)

Click here for additional data file.

13 Sep 2020

Dear Dr Chan,

Thank you very much for submitting your manuscript "SARS-CoV-2 lineage B.6 is the major contributor to transmission in Malaysia" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

Your manuscript has been reviewed by three experts in the field. Reviewers have made some major suggestions for improvement. Please address all comments from all reviewers. In particular, you are strongly advised to perform in depth analyses of other genomic regions of the SARS-CoV-2 from different lineages and highlight the importance of this study towards the pandemic as suggested by reviewers.

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Susanna Kar Pui Lau

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Your manuscript has been reviewed by three experts in the field. Reviewers have made some major suggestions for improvement. Please address all comments from all reviewers. In particular, you are strongly advised to perform in depth analyses of other genomic regions of the SARS-CoV-2 from different lineages and highlight the importance of this study towards the pandemic as suggested by reviewers.

I am willing to consider your revised manuscript that addresses all the suggestions and criticisms of the reviewer. The modified version of your paper may be sent back to the original reviewers prior to its possible acceptance. We cannot, of course, promise publication at that time.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: It is a SARS-CoV-2 retrospective genomic epidemiological study and the authors appear to have taken necessary steps to elucidate their findings as such.

Some clarification/changes regarding the sample sizes is required.

Reviewer #2: The objective of the study is clearly presented, the study design is appropriate, the population has been described. However, the sample size is limited compared to the confirmed cases. There are no ethical concerns. No particular statistics analysis have been used.

Reviewer #3: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Yes

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The results require some clarification/changes. There are no major errors in the analysis.

Reviewer #2: The analysis matches the plan. The results are missing some information and also a new schematic chart would be helpful to better understand where the mutations are located.

Reviewer #3: YES

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The conclusions need some clarification/changes and there is acknowledgement of the limitations of the sequencing studies.

Reviewer #2: No limitations are mentioned. The conclusions reflect the data. However, the authors should highlight the importance of this data in the pandemic, how these data advance the global understanding of SARS-CoV-2 mechanisms of infections, spreading and lethality.

Reviewer #3: YES

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: A new schematic chart would be helpful to better understand where the mutations are located.

With this new chart specific for the mutations, the current trees should be changed.

Reviewer #3: NONE

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The authors describe a SARS-CoV-2 genomic epidemiology study that took place in Malaysia and the finding of a specific viral lineage that is circulating in SEA regions.

They also find that a certain mutation leads to reduced sensitivity in commercial assays for the virus.

Thanks to the authors to provide all necessary details to gauge the sequencing and analysis protocols used in the study.

It would be very helpful if the significance of the study to researchers and clinicians/diagnostics can be described in greater detail (Abstract and Introduction).

"In particular, we were interested in determining the role of the Tablighi Jamaat gathering in regional spread."

Were the 58 patients from the religious gathering?

In the Methodology, explicit details are needed on how many of the 58 patients were from the religious gathering in Malaysia.

And how many of the other 50 Malaysian sequences were from the religious gathering? (Is it 39?)

The total number of B.6 sequences is not consistent between the last paragraph of Methodology and 3rd paragraph of Results.

A detailed split of B.6 sequences can be provided as: sequenced at UMMC + sequenced at other centers in Malaysia + sequenced at other global centers.

Regarding the events that took place and the statement, "of other Malaysian sequences in GISAID dating from the Tablighi Jamaat gathering".

In the above statement, the timeline of events are not clear.

Also, in the recently published work of the authors "Complete Genome Sequences of SARS-CoV-2 Strains Detected in Malaysia",

Fig. 1A the religious event is dated as 29 Feb - 3 Mar, but in the current study the date is 27 Feb - 1 Mar.

Thus, the authors should describe with detail and certainty about the events that took place and that the sequences were from infections originating at the religious gathering.

Rearranging some final sections of the Methodology and beginning of Results could be helpful.

Fig.1: If there are 39 sequences from the religious gathering why are only 3 highlighted?

To avoid blocking the dendrogram, moving the "red point" to the right side of the sequences would be better.

Fig. 3A: Onlyan outline of the histogram could be sufficient. This would also make the lineage markers more clear to see.

Fig. 3C: Would be helpful to include legend in the figure for Southeast Asian country bars vs other country bars

Reviewer #2: The manuscript “SARS-CoV-2 lineage B.6 is the major contributor to transmission in Malaysia” describes the predominant spreading of the B.6 lineage in Malaysia after a religious mass gathering in Kuala Lumpur. This event is associated with the main wave of COVID-19 cases in Malaysia. The authors analyzed 58 genomes sequences from COVID-19 patients that attended the religious event in Kuala Lumpur and 50 Malaysian sequences available in the GISAID database. The authors detected 9 different lineages, and among these, the B.6 became the predominant cause of community transmission in Malaysia after the likely introduction during the religious mass gathering.

The references are appropriate.

Major revisions:

- The authors use sometimes SARS-CoV-2 and COVID-19 as synonymous. They are not: SARS-CoV-2 is the novel coronavirus, while COVID-19 includes all the medical conditions caused by SARS-CoV-2 (as also correctly stated by the authors in the introduction). I would suggest the authors to review the manuscript and use the correct terminology.

- The authors should consider adding a more in-depth analysis of the sequences and their mutations. The authors just mentioned a mutation in ORF3a, one in the spike protein, and few more. What about the other viral genes? For example, the one in the polymerase mentioned by Pachetti et al, J Transl Med 18, 179 (2020)?

- A schematic chart would be helpful to better understand where the mutations are located.

- A few typos need to be corrected by the authors.

Reviewer #3: This is a well written manuscript demonstrating how genetic analysis can inform about transmission dynamics of SARS-CoV-2.

The study shows very nicely how a religious gathering fuelled the spread of the B.6 lineage in Malaysia. The results are clearly presented and my only comment to the results section is Fig. 3A where I would prefer to see the results as rates for instance per 100 000 population and not actual number of cases.

In the discussion I wonder if the authors could elaborate on whether the B.6 had a higher or lower mortality compared to other lineages?

--------------------

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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13 Oct 2020

Submitted filename: Response to reviewer.pdf

Click here for additional data file.

30 Oct 2020

Dear Dr Chan,

We are pleased to inform you that your manuscript 'SARS-CoV-2 lineage B.6 is the major contributor to transmission in Malaysia' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Susanna Kar Pui Lau, M.D.

Deputy Editor

PLOS Neglected Tropical Diseases

Susanna Kar Pui Lau

Deputy Editor

PLOS Neglected Tropical Diseases

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: This is a revised manuscript.

Reviewer #2: The authors updated the numbers and figures based on the data available up to September 20th 2020. The authors have taken the necessary steps to describe their findings.

There are no ethical concerns.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: The results have been better clarified. Table S3 has been added.

There are no major errors in the analysis.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: The conclusions have been improved and clarified.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The authors have addressed all the comments sufficiently.

Reviewer #2: The authors provided more details regarding the samples and the results.

A more in deep analysis of the sequences has been conducted.

minor revisions:

- the legends for figure 1-2-3 are missing.

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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.

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Reviewer #1: No

Reviewer #2: No

20 Nov 2020

Dear Dr Chan,

We are delighted to inform you that your manuscript, "SARS-CoV-2 lineage B.6 is the major contributor to early pandemic transmission in Malaysia," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases