Journal:Comprehensive analyses of SARS-CoV-2 transmission in a public health virology laboratory

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Full article title Comprehensive analyses of SARS-CoV-2 transmission in a public health virology laboratory
Journal Viruses
Author(s) Zuckerman, Neta S.; Pando, Rakafet; Bucris, Efrat; Drori, Yaron; Lustig, Yaniv; Erster, Oran;
Mor, Orna; Mendelson, Ella; Mandelboim, Michael
Author affiliation(s) Chaim Sheba Medical Center, Israel Ministry of Health, Tel-Aviv University
Primary contact Email: michalman at sheba dot health dot gov dot il
Year published 2020
Volume and issue 12(8)
Article # 854
DOI 10.3390/v12080854
ISSN 1999-4915
Distribution license Creative Commons Attribution 4.0 International
Website https://www.mdpi.com/1999-4915/12/8/854/htm
Download https://www.mdpi.com/1999-4915/12/8/854/pdf (PDF)
Emblem-important-yellow.svg This article should be considered a work in progress and incomplete. Consider this article incomplete until this notice is removed.

Abstract

SARS-CoV-2 has become a major global concern as of December 2019, particularly affecting healthcare workers. As person-to-person transmission is airborne, crowded closed spaces have had high potential for rapid virus spread, especially early in the pandemic when social distancing and mask wearing were not mandatory. This retrospective study thoroughly investigates a small-scale SARS-CoV-2 outbreak in Israel’s central virology laboratory (ICVL) in mid-March 2020, in which six staff members and two related family members were infected. Suspicions regarding infection by contaminated surfaces in ICVL facilities were nullified by the negative results of a SARS-CoV-2 real-time polymerase chain reaction (qPCR) analysis of swiped work surface samples. Complete SARS-CoV-2 genomes were sequenced, and mutation analyses showed inclusion of all samples to clades 20B and 20C, possessing the spike mutation D614G. Phylogenetic analysis clarified transmission events, confirming S1 as having infected at least three other staff members while refuting the association of a staff member’s infected spouse with the ICVL transmission cluster. Finally, serology tests exhibited IgG and IgA antibodies in all infected individuals and revealed the occurrence of asymptomatic infections in additional staff members. This study demonstrates the advantages of molecular epidemiology in elucidating transmission events and exemplifies the importance of good laboratory practice, physical distancing, and mask wearing in preventing SARS-CoV-2 spread, specifically in healthcare facilities.

Keywords: 2019-nCoV, SARS-CoV-2, COVID-19, staff, infection, next-generation sequencing (NGS)

Introduction

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel coronavirus that emerged in Wuhan, China in December 2019[1] and has rapidly spread across China and to many countries worldwide, causing severe respiratory disease (COVID-19) leading to substantial morbidity and mortality.[2][3][4][5][6] This novel virus is a potential threat to human health worldwide and a major global health concern due to person-to-person transmission, a current lack of vaccination, and a lack of effective therapeutic options.[3][7] Major SARS-CoV-2 worldwide clades have been proposed by nomenclature systems, including Nextstrain[8] and the Global Initiative on Sharing All Influenza Data (GISAID).[9] These are based on viral genomes from >57,000 sequences submitted in GISAID.[9] For example, using Nextstrain’s nomenclature, there are currently five major clades: 19A (the root clade) and 19B, and clades 20A, B, and C. These are widespread in Europe and include a mutation in the spike protein, D614G, that is associated with increased infectivity and higher viral loads.[10]

Non-SARS-CoV-2 human coronaviruses have been circulating worldwide since the late 1960s.[11][12] The current rate of circulation of SARS-CoV-2 in Israel in the winter season is still unknown; however, analysis of Israeli specimens during the 2015–2016 winter season revealed that non-SARS-CoV-2 human coronaviruses circulate simultaneously with other common respiratory viruses, with 10% human coronavirus-positive cases.[13]

SARS-CoV-2 circulation in the general population in Israel and worldwide is being assessed using real-time polymerase chain reaction (qPCR). A rapid development of qPCR diagnostic tests specific for SARS-CoV-2 genes has enabled fast and accurate laboratory tests for suspected individuals.[14] These tests were successfully evaluated in Israel’s central virology laboratory (ICVL), where SARS-CoV-2 suspected specimens were exclusively examined, starting from the first importation case of SARS-CoV-2 into Israel at the end of February until the middle of March 2020. Starting with the first suspected case in Israel, all specimens received in ICVL facilities were dealt with using the strictest safety directions and [[Biosafetly level#Levels|biosafety level 2 (BSL2) or greater safety conditions.[15][16] Until mid-March 2020, all SARS-CoV-2 positive cases in Israel were isolated in a designated quarantine facility; however, physical distancing and mandatory mask-wearing were not customary or enforced at that time in Israel.

In mid-March 2020, several cases of SARS-CoV-2 infection were identified in ICVL, some of which probably originated from an infected worker, as speculated by the inquiry-based epidemiological investigation. SARS-CoV-2 airborne transmission was demonstrated to be the most efficient among all transmission routes[17][18], and contagious even in the pre-symptomatic stages[19][20], such that silent virus spread easily occurs. Infection at workplaces was shown as a common transmission route in Israel in the early stages of the virus' spread, probably facilitated, in the case of the ICVL outbreak, by crowded workspaces and lack of social distancing and mask wearing at that time.

This study thoroughly investigates the SARS-CoV-2 ICVL outbreak by examining infected ICVL workers, several epidemiologically-related family members, and work surfaces from ICVL facilities. Application of SARS-CoV-2 whole genome next-generation sequencing (NGS), qPCR, serology testing, and phylogenetic tree analyses elucidate person-to-person transmission events, map individual and common mutations, and examine suspicions regarding contaminated surfaces. This study demonstrates the added value of molecular epidemiology based on complete viral genomes in elucidating person-to-person transmission, reveals silent infections in non-symptomatic ICVL staff members via serology testing, and confirms that the strict safety regulations observed in ICVL most likely prevented further spread of the virus.

Materials and methods

Specimen collection, nucleic acid extraction, and viral genome quantification by qPCR

Immediately following the identification of the first ICVL infection case (S1) on March 15, 2020, nasopharyngeal swabs from all 56 ICVL staff members and another ten non-ICVL staff who worked at the lab around this time were collected, most of them on the same day and a few on the next day. This comprehensive screening test was performed only once. Additional tests for ICVL staff were conducted for a symptomatic individual (n = 1), symptomatic relatives (n = 2), and for essential workers who were required to work at the laboratory (n = 2). Viral genomes were extracted from 200 µL respiratory specimens with the MagNA PURE 96 (Roche, Mannheim, Germany) according to the manufacturer instructions, and real-time (or quantitative) reverse transcription polymerase chain reaction (qRT-PCR) reactions using primers corresponding to the SARS-CoV-2 envelope (E) gene were performed as previously described by Corman et al.[14] All specimens were tested for the human RNAseP gene, which served as a housekeeping gene. The qRT-PCR reactions were performed in 25 µL Ambion Ag-Path Master Mix (Life Technologies, Carlsbad, CA, USA) using TaqMan Chemistry on the ABI 7500 instrument. Nucleic extraction specimens from SARS-CoV-2-positive staff members (S1–S6) and related family members (S7—S4′s spouse and S8—S3′s spouse) were taken for further molecular analysis.

Specific amplification of SARS-CoV-2 from clinical specimens

RNA in extracted nucleic acids was reverse transcribed to single strand cDNA using SuperScript IV (ThermoFisher Scientific, Waltham, MA, USA) as per manufacturer’s instructions. SARS-CoV-2-specific primers designed to capture SARS-CoV-2 whole genome (version 1—total 218 primers, divided into two primer pools designed by Josh Quick from ARTIC Network) were used to generate double strand cDNA and amplify it via PCR using Q5 Hot Start DNA Polymerase (NEB).[21] Briefly, each specimen underwent two PCR reactions with primer pool 1 or 2 and 5X Q5 reaction buffer, 19 mM dNTPs and nuclease-free water. Resulting DNA was combined and quantified with the Qubit dsDNA BR Assay kit (ThermoFisher Scientific) as per manufacturer’s instructions, and 1ng of amplicon DNA in 5 µL per specimen was taken into library preparation.

Library preparation and sequencing

Libraries were prepared using the NexteraXT library preparation kit and NexteraXT index kit V2 as per manufacturer’s instructions (Illumina, San Diego, CA, USA). Libraries were purified with AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA), and library concentration was measured by Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Library validation and mean fragment size was determined by TapeStation 4200 via DNA HS D1000 kit (Agilent, Santa Clara, CA, USA). The mean fragment size was ~400 bp, as expected. The library mean fragment size and concentration molarity was calculated and each library was diluted to 4 nM. Libraries were pooled, denatured, and diluted to 10pM and sequenced on MiSeq with V3 2X300 bp run kit (Illumina). Sequences are available in GISAID (accession numbers: EPI_ISL_435284, EPI_ISL_435286, EPI_ISL435287, EPI_ISL435289, EPI_ISL435291, EPI_ISL_435292, EPI_ISL447250, EPI_ISL447251).

Bioinformatics analyses

The fastq files were subjected to quality control using FastQC[22] and MultiQC[23], and low-quality sequences were filtered using trimmomatic.[24] To obtain a consensus sequence per specimen, paired-end fastq files were combined for each specimen via the Unix cat command. SARS-CoV-2 reference genome was downloaded from the national center for biotechnology information (NCBI) (NC_045512.2) and indexed using Burrows-Wheeler aligner (BWA).[25] Combined fastq files were mapped to the indexed reference genome using BWA mem.[25] SAMtools suite[26] was used to convert sam to bam files, remove duplicates, and filter unmapped reads. The bam files were sorted, indexed, and subjected to quality control using SAMtools suite. Coverage and depth of sequencing was calculated from sorted bam files using a custom Perl script. Integrative genome viewer (IGV) was used to observe sequencing coverage per position along the genome.[27] A consensus sequence was constructed for each specimen using SAMtools mpileup and bcf tools[28] and converted to a fasta file using seqtk.[29]

Multiple alignment of the sequences with the NC_045512.2 reference and additional sequences was carried out using clustal omega.[30]

For the phylogenetic tree construction, the general time reversible model with proportion of invariable sites and gamma plus invariant site-distributed rate heterogeneity (GTR + G + I model) was chosen using jModelTest 2.[31] The phylogenetic tree was constructed and visualized via MEGA7[32] using the maximum likelihood method, with 1000 bootstrap runs.

Clade annotations were attained from Nextstrain[8], who identify variants that define clades of interest from sequences submitted to GISAID by labs worldwide and updates them periodically in clades.tsv.[33]

Additional bioinformatic analyses such as translation from nucleotide to amino acid sequences, comparison of differences across sequences, and sample clustering were conducted and visualized using R and Bioconductor packages Seqinr[34], HDMD[35], and ggplot2.[36] Classification to amino acid groups was set according to physiochemical attributes determined by Atchley et al.[37]

Wipe test sampling

Immediately following the identification of the first ICVL infection case (S1), six environmental samples (as detailed in Table 1) were obtained from all SARS-CoV-2 relevant surfaces and equipment in the ICVL using into ∑-Virocult (Medical Wire, Corsham, UK) virus transport medium as described by Bright et al.[38] Three of the samples were taken from the biosafety cabinets (BSCs) in which all suspected SARS-CoV-2 were opened, while the other three samples were obtained from all other surfaces in these facilities, including door knobs, the outer surface of all equipment in the room, etc., with special attention to “high-touched areas.” All samples were tested for SARS-CoV-2 using qRT-PCR as mentioned above.


Table 1. Wipe test sampling plan
Sample # Facility Sampled equipment
1 Specimen reception area All work surfaces and equipment in the room including knobs, chairs, doors, etc.
2 Specimen reception area Biosafety cabinets’ (BSC) outside and inside surface
3 Specimen sampling room #1 All work surfaces and equipment in the room including knobs, chairs, doors, etc.
4 Specimen sampling room #1 BSC outside and inside surface
5 Specimen sampling room #2 All work surfaces and equipment in the room including knobs, chairs, doors, etc.
6 Specimen sampling room #2 BSC outside and inside surface

Serology

IgG and IgA antibodies against SARS-CoV-2 were detected by an in-house ELISA using antigen prepared as described by Amanat et al.[39] For the ELISA, a 96-well microtiter Polysorb plate (Nunc, Thermo, Roskilde, Denmark) was coated overnight at 4 °C with 1 µg/mL of RBD antigen for detection of IgG and 2 µg/mL for detection of IgA antibodies. After blocking with 5% skimmed milk at 25 °C for 60 minutes, human serum samples (diluted 1:100 with 3% skimmed milk), were added to antigen coated wells. The plate was incubated at 25 °C for 120 minutes, washed, and goat anti-human IgG horseradish peroxidase (HRP) conjugate (Jackson ImmunoResearch, Philadelphia, PA, USA) (diluted 1:15,000) or anti human IgA HRP conjugate (Abcam, Cambridge, MA, USA) was added to each well for 60 minutes. After addition of TMB substrate and stop solution (1 M HCl), the OD of each well was measured at 450 nm. An ELISA index value below 0.9 was considered negative, between 0.9 and 1.1 considered equivocal and equal, and above 1.1 considered positive. In a validation study which included 633 serum samples obtained from 309 persons infected by SARS-CoV-2 and 324 healthy, uninfected individuals, specificity and sensitivity of the IgG was 98% and 88%, respectively and specificity and sensitivity of the IgA was 98% and 80%, respectively.

Results

SARS-CoV-2-specific qRT-PCR assay identifies SARS-CoV-2 positive ICVL staff and relatives

Following confirmed SARS-CoV-2 in ICVL staff member S1, SARS-CoV-2 specific qRT-PCR assays were conducted for all ICVL staff and relevant family members on March 15, 2020 and on the next day. Only two additional staff members tested positive for SARS-CoV-2 (Table 2): S2, with a relatively high cycle threshold (Ct) (Ct = 33.07) and S3 with a much lower Ct (Ct = 18.77). A few days later, on March 23, two additional ICVL staff members S4 and S6 tested positive for SARS-CoV-2 (Ct = 26 and 22 respectively). On March 29, an additional qRT-PCR assay for all self-isolated ICVL staff was conducted as a precaution test prior to their return to the lab. Surprisingly, an additional ICVL staff member, S5, tested positive (Ct = 28.58) albeit non-symptomatic.

Table 2. Infected cases characteristics and sequencing parameters. Characteristics for each case include SARS-CoV-2 cycle threshold (Ct), age, date of detection and estimated date of infection according to the epidemiological investigation, and association with the Israel Central Virology Laboratory (ICVL) transmission chain. Sequencing characteristics include the total number of sequence reads mapped to SARS-CoV-2 reference (# mapped reads), percent of SARS-CoV-2 genome covered by the sequence reads (% coverage), and the average depth of sequencing per sample. * = Estimated date of infection according to the epidemiological investigation
Case characteristics Sequencing parameters
Specimen # SARS-CoV-2 Ct IgG/IgA values Age Date of detection Estimated date of infection* Transmission chain # of mapped reads % coverage Average depth
S1 14.30 3.36/3.34 55 15.3.20 unknown ICVL 3,806,897 100.00 6095
S2 33.07 4.87/13.86 65 15.3.20 unknown NA 5,096,580 98.00 5357
S3 18.77 4.52/5.9 46 15.3.20 10.3.20 NA 3,495,706 99.65 5501
S4 26.00 4.77/6.77 39 23.3.20 14.3.20 ICVL 1,739,690 99.24 4713
S5 28.58 4.71/1.83 61 29.3.20 14.3.20 ICVL 1,419,044 99.96 4958
S6 22.00 4.75/1.76 41 23.3.20 14.3.20 ICVL 3,380,868 99.90 6014
S7 24.00 6.69/2.1 41 29.3.20 14.3.20 NA 8,580,675 99.99 40,466
S8 22.00 7.17/10.23 52 18.3.20 10.3.20 NA 9,498,576 100.00 45,041

The spouse of S3 (S8) presented with symptoms post-exposure to a verified SARS-CoV-2 individual, and was therefore examined and found positive on March 18 (Ct = 22). The spouse of S4 (S7) also presented with symptoms and was found positive on March 29 (Ct = 24). All infected individuals were in an age range of 39–65 and were non-symptomatic or exhibited mild symptoms (Table 2). An inquiry-based epidemiological investigation was conducted for each infected individual (data not shown), and estimated dates of infection were inferred. Definitive information regarding transmission was not attainable for each worker or related family member. Nevertheless, within the ICVL transmission chain, patient S1 was determined as having infected multiple individuals according to the epidemiological investigation.

Whole genome sequencing-based molecular epidemiology elucidates transmission events

To molecularly infer transmission within the ICVL local outbreak, SARS-CoV-2 was amplified in all SARS-CoV-2 positive specimens from ICVL staff and related family members using specific primers, and whole genomes were sequenced from each specimen via Illumina technology. All specimens had >99% coverage and an average sequencing depth of 13,583, with the exception of S2 which had 98% coverage, probably due to a high cycle threshold (Ct) value (i.e., lower virus quantity) (Table 2).

A phylogenetic tree was constructed to depict relationships amongst all specimens. All specimens sequenced were associated with clade 20, harboring the clade’s mutations A23403G and C14408T (Nextstrain nomenclature[8]). All ICVL staff members exhibited the 20C clade-defining mutations G25563T and C1059T. S8 (S3′s spouse) lacked one of the 20C mutations C1059T. Interestingly, S7 (S4′s spouse) exhibited three additional mutations—G28881A, G28882A and G28883C—which are associated with clade 20B. S1, S4, and S5 had identical sequences (Figure 1), while S6 exhibited only one difference: a uniquely observed mutation in nsp12—G15243T (Figure 1 and Table 3). Given that an inquiry-based epidemiological investigation determined S1′s date of infection to an earlier time compared to S4, S5, and S6 (Table 2), these results confirm that S1 infected at least three other ICVL workers (S4, S5, and S6). Two additional ICVL members, S2 and S3, exhibited two and one differences, respectively, compared to the identical S1, S4, and S5 sequences (Table 3), and additional mixed-nucleotide differences characteristic of a quasi-species (Table S1). Although they shared the 20C clade-defining mutation C1059T with ICVL members S1, S4, and S5, which produced a high-confidence split from non-ICVL members S7 and S8 (bootstrap value of 73%, Figure 1), given their additional mutations, which may have been acquired individually or from a different transmission chain, S2 and S3 cannot be placed within the ICVL transmission chain. S8 was placed in the 20C clade due to a shared mutation with all ICVL members, G25563T, in addition to its own unique mutation. However, S7 was placed outside of the ICVL transmission chain due to two mutations from a related but different clade, 20B. These conclusions are further supported by a larger-scale phylogenetic tree including the ICVL workers and family member specimens, in addition to 25 randomly chosen Israel-based specimens downloaded from GISAID, wherein S7 is placed with non-ICVL specimens with 88% confidence (Figure S1).


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  34. ↑ Charif, D.; Lobry, J. (2007). "SeqinR 1.0-2: a contributed package to the R project for statistical computing devoted to biological sequences retrieval and analysis". In Bastolla, U.; Porto, M.; Roman, H. et al.. Structural approaches to sequence evolution: Molecules, networks, populations. Biological and Medical Physics, Biomedical Engineering. Springer Verlag. pp. 207–32. ISBN 9783540353058. 
  35. ↑ McFerrin, L. (27 February 2013). "HDMD: Statistical Analysis Tools for High Dimension Molecular Data (HDMD)". The Comprehensive R Archive Network. https://cran.r-project.org/web/packages/HDMD/index.html. 
  36. ↑ Wickham, H. (2016). ggplot2 (2nd ed.). Springer. ISBN 9783319242774. 
  37. ↑ Atchley, W.R.; Terhalle, W.; Dress, A. (1999). Positional dependence, cliques, and predictive motifs in the bHLH protein domain. 48. pp. 501-16. doi:10.1007/pl00006494. PMID 10198117. 
  38. ↑ Bright, K.R.; Boone, S.A.; Gerba, C.P. (2010). Occurrence of bacteria and viruses on elementary classroom surfaces and the potential role of classroom hygiene in the spread of infectious diseases. 26. pp. 33–41. doi:10.1177/1059840509354383. PMID 19903773. 
  39. ↑ Amanat, F.; Stadlbauer, D.; Strohmeier, S. et al. (2020). A serological assay to detect SARS-CoV-2 seroconversion in humans. 26. pp. 1033–36. doi:10.1038/s41591-020-0913-5. PMID 32398876. 

Notes

This presentation is faithful to the original, with only a few minor changes to presentation. In some cases important information was missing from the references, and that information was added.

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