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Abril, Cristina Andrés, Aroa Silgado, Alexandra Navarro, Anna Suy, Elena Sulleiro, Tomàs Pumarola, Josep Quer, Andrés Antón PII: S1198-743X(21)00382-7 DOI: https://doi.org/10.1016/j.cmi.2021.07.008 Reference: CMI 2615 To appear in: Clinical Microbiology and Infection Received Date: 24 February 2021 Revised Date: 28 June 2021 Accepted Date: 5 July 2021 Please cite this article as: Piñana M, Abril JF, Andrés C, Silgado A, Navarro A, Suy A, Sulleiro E, Pumarola T, Quer J, Antón A, Viral populations of SARS-CoV-2 in upper respiratory tract, placenta, amniotic fluid and umbilical cord blood support viral replication in placenta, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2021.07.008. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2021 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved. 1 Viral populations of SARS-CoV-2 in upper respiratory tract, placenta, 2 amniotic fluid and umbilical cord blood support viral replication in 3 placenta 4 Maria Piñana1, Josep F. Abril2, Cristina Andrés1, Aroa Silgado1, Alexandra Navarro3, 5 Anna Suy4, Elena Sulleiro1, Tomàs Pumarola1, Josep Quer1,5*a, Andrés Antón1a. 1 6 Microbiology Department, Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron of 7 Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain ro 2 8 Computational Genomics Lab; Genetics, Microbiology and Statistics Department; 9 -p Institut de Biomedicina (IBUB); Universitat de Barcelona; Barcelona, Catalonia, Spain re 3 10 Pathology Department, Hospital Universitari Vall d’Hebron, Barcelona, Spain lP 4 11 Maternal Fetal Medicine Unit, Department of Obstetrics, Hospital Universitari Vall na 12 d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain ur 5 13 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas Jo 14 (CIBERehd); Instituto de Salud Carlos III; Madrid, Spain 15 aAuthor Josep Quer and author Andrés Antón were co-principal investigators. 16 *Corresponding author: Josep Quer 17 Liver Unit, Liver Diseases - Viral Hepatitis 18 Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Hospital Universitari 19 Vall d’Hebron Barcelona Hospital Campus, Passeig Vall d’Hebron 119-129, 08035, 20 Barcelona, Spain 1 21 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas 22 (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain 23 E-mail: Josep.quer@vhir.org 24 Running title: SARS-CoV-2 populations in non-respiratory tissues. 25 Keywords: SARS-CoV-2, quasispecies, placenta, amniotic fluid, blood cord, pregnancy, 26 vertical transmission. 27 Length main text: 794 of ro -p re lP na ur Jo 2 28 MAIN TEXT 29 To the editor, 30 In the current context of SARS-CoV-2 pandemic, one of the main concerns is whether 31 SARS-CoV-2 can be vertically transmitted. In addition to conventional testing for SARS- 32 CoV-2 detection in respiratory specimens, the study of viral populations contributes to 33 elucidate the infection dynamics. of 34 A woman at 40 weeks and 4 days of pregnancy was admitted to our hospital at the ro 35 first stage of labour. Since she had presented cough and malaise for 6 days, and her 36 -p living relatives had tested positive for SARS-CoV-2, a naso/oropharyngeal swab was re 37 collected. Amniotic fluid before rupture of membranes, placenta and umbilical cord lP 38 blood samples were collected. Although the newborn remained asymptomatic, a na 39 nasopharyngeal aspirate, serum and peripheral blood were collected at birth, and an 40 additional serum six weeks later. Detection of SARS-CoV-2 by real-time RT-PCR assays ur 41 and serological testing were performed. The placenta was studied by histology, Jo 42 immunohistochemistry and in situ hybridization (ISH). Institutional Review Board 43 approval (PR(AG)259/2020 and PR(AMI)181/2020) was obtained from the HUVH 44 Clinical Research Ethics Committee. WGS of SARS-CoV-2 was performed following the 45 ARTIC protocol (https://artic.network/ncov-2019) and sequenced with MiSeq 46 (Illumina, USA) (1). Bioinformatic analyses were run using FastQC, Trinity, lofreq, and 47 Pangolin v2.0.7 (2) among others. 48 SARS-CoV-2 was laboratory-confirmed in all maternal samples. The lowest Ct value was 49 observed in placenta (21,91-23,7), indicative of a higher viral load than in upper 3 50 respiratory tract (URT; 25,26-28,7). This may suggest that either the virus has 51 replicated in this tissue or that viral load in nasopharynx had already decreased after 6 52 days of symptoms. Both the fact that the virus was also present in the amniotic fluid, 53 whose sampling was in sterile conditions prior to the rupture of membranes, and the 54 fact that SARS-CoV-2 was found in the cytoplasm of the placenta’s trophoblastic cells, 55 support compartmentalized SARS-CoV-2 replication in placenta. Newborn’s respiratory 56 and serum samples were SARS-CoV-2-negative at birth time; IgG and IgA were of 57 detected at six weeks of age, suggesting a probable post-partum infection from the ro 58 mother. 59 -p Viral consensus sequences from the four maternal tissues were identical, carrying re 60 D614G in the Spike, a 9-nucleotide deletion (686-694) in nsp1, and two silent lP 61 mutations (241C>T in 5’UTR and 3,037C>T in nsp3). PANGOLIN analyses revealed they na 62 belonged to B.1.5 lineage, which was the most prevalent in Europe at that time (3). ur 63 Minor viral variants (MVV) were mostly present at <2% frequency (Figure 1). The Jo 64 number of variants was higher in URT (677), though a noteworthy presence of MVV 65 was observed in placenta (233) and amniotic fluid (330). The coverage of the umbilical 66 cord blood was low and could not be compared. To our knowledge, this is the first 67 description of quasispecies in non-respiratory specimens. The URT had a 60% of 68 unique MVV, while placenta and amniotic fluid presented a 30% each. Though sharing 69 a considerable number of mutations, the profile of variants in placenta was different 70 from the nasopharyngeal swab’s, suggesting that there might be compartmentalized 71 virus replication (4), compatible with viral replication in placenta and supported by the 72 high viral load. Most mutations (53%; 510/956) had a high impact in the protein, 4 73 adding or replacing stop codons or causing frameshifts. Notably, there were large 74 indels throughout all the genome involving up to 48 nucleotides. These indels were 75 detected at very low frequencies and were mostly observed in nsp3, nsp12 and Spike. 76 These two nsp have not been deeply studied yet, but minor viral deletions in Spike 77 have already been observed (1). Interestingly, in placenta and nasopharyngeal 78 epithelium, but not in amniotic fluid, MVV carrying genetic deletions were detected 79 upstream, very close to the S1/S2 cleavage site where naturally gene deletions were of 80 previously reported in mild and severe patients at low frequencies as a viral ro 81 attenuation mechanism of infection (1). 82 -p This study presents some limitations. Only one patient was included, and more re 83 patients should be monitored in further studies to confirm these findings. Also, lP 84 maternal blood collected at the time of labour could not be studied to confirm or na 85 reject RNAemia, even though this is usually related to more severe cases. ur 86 This study provides further evidence that SARS-CoV-2 can replicate in placenta and Jo 87 cross the placenta barrier to the amniotic fluid. Detecting SARS-CoV-2 in the amniotic 88 fluid shows that the virus can cross the placenta barrier. Moreover, different 89 quasispecie composition between maternal respiratory and non-respiratory 90 specimens, as well as a high viral load and histological finding in placenta suggests that 91 the virus can arrive to placenta, where the virus can replicate. More observational 92 studies with larger number of patients must be done to confirm this replication in the 93 intrauterine environment of the foetus, as well as to corroborate if maternal 94 antibodies could be preventing vertical transmission. One recommendation from this 5 95 study is the close monitoring of SARS-CoV-2 infection during pregnancy, as done with 96 other potential congenital pathogens (TORCH). 97 Authors’ contributions 98 MP and CA contributed in the conception and design of the work, the analysis and 99 interpretation of data and drafted and revised the manuscript. JFA contributed in the 100 bioinformatic analysis and interpretation of data, and drafted and revised the 101 manuscript. ASi contributed with the acquisition and analysis of data and revised the of 102 manuscript. AN contributed with all the histopathological analyses and interpretation ro 103 of data, and drafted and revised the manuscript. ASu contributed with the sampling 104 -p and acquisition of the patient’s data, and drafted and revised the manuscript. ES, TP, re 105 JQ and AA contributed in the conception and design of the work and drafted and lP 106 revised the manuscript. All authors approved the submitted version. na 107 Acknowledgments ur 108 This study was supported by the Direcció General de Recerca i Innovació en Salut Jo 109 (DGRIS) Catalan Health Ministry Generalitat de Catalunya through Vall d’Hebron 110 Institut de Recerca (VHIR); European Development Regional Fund (ERDF) “A way to 111 achieve Europe” by Spanish Network for Research in Infectious Diseases [REIPI 112 RD16/0016/0003]; and Centro para el Desarrollo Tecnológico Industrial (CDTI) from 113 the Spanish Ministry of Economy and Business [grant number IDI-20200297]. JFA 114 benefits from Generalitat de Catalunya (AGAUR) grant [grant number 2017SGR-1455]. 115 The funder had no role in the study design, data collection and analysis, decision to 116 publish, or manuscript preparation. We gratefully acknowledge the authors, 6 117 originating and submitting laboratories of the sequences from GISAID’s EpiCov™ 118 Database on which this research is based. 119 Competing interests 120 The authors declare that they have no competing interests. 121 References 122 1. Andrés C, Garcia-Cehic D, Gregori J, Piñana M, Rodriguez-Frias F, Guerrero- of 123 Murillo M, Esperalba J, Rando A, Goterris L, Codina MG, Quer S, Martín MC, ro 124 Campins M, Ferrer R, Almirante B, Esteban JI, Pumarola T, Antón A, Quer J. 125 -p 2020. Naturally occurring SARS-CoV-2 gene deletions close to the spike S1/S2 re 126 cleavage site in the viral quasispecies of COVID19 patients. Emerg Microbes lP 127 Infect 9:1900–1911. na 128 2. Rambaut A, Holmes EC, O’Toole Á, Hill V, McCrone JT, Ruis C, du Plessis L, Pybus ur 129 OG. 2020. A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist Jo 130 genomic epidemiology. Nat Microbiol 5. 131 3. Alm E, Broberg EK, Connor T, Hodcroft EB, Komissarov AB, Maurer-Stroh S, 132 Melidou A, Neher RA, O’Toole Á, Pereyaslov D. 2020. Geographical and 133 temporal distribution of SARS-CoV-2 clades in the WHO European Region, 134 January to June 2020. Eurosurveillance 25:1–8. 135 4. Rueca M, Bartolini B, Gruber CEM, Piralla A, Baldanti F, Giombini E, Messina F, 136 Marchioni L, Ippolito G, Di Caro A, Capobianchi MR. 2020. Compartmentalized 137 replication of SARS-CoV-2 in upper vs lower respiratory tract assessed by whole 7 138 genome quasispecies analysis. Microorganisms 8:1–12. 139 of ro -p re lP na ur Jo 8 140 Figure legends 141 Figure 1. Representation of minor viral variants along the genome in each sample. 142 The X-axis represents the in-scale SARS-CoV-2 genome and the Y-axis represents the 143 frequency of minor viral variants. Black bars represent minor viral variants which are in 144 two or more tissues, while pink bars represent unique mutations. of ro -p re lP na ur Jo 9 Jo ur na lP re -p ro of