key: cord-0699595-re02j03h
authors: ESSAIDI-LAZIOSI, M.; Perez Rodriguez, F. J.; Sattonnet Roche, P.; Hulo, N.; Jacquerioz, F.; Kaiser, L.; Eckerle, I.
title: Assessment of the infectious threshold of SARS-CoV-2 in primary airway epithelial cells
date: 2021-05-19
journal: nan
DOI: 10.1101/2021.05.18.21257110
sha: e08201366db1bb3e20471b077c4e459283cecc3a
doc_id: 699595
cord_uid: re02j03h

Comparison of virus isolation success from clinical samples across a range of viral loads inoculated in parallel on Vero E6 and human airway epithelia (HAE) showed lower success of virus isolation in HAE, suggesting an overestimation of actual infectiousness in humans using Vero E6 cell lines, commonly considered as reference.

Comparison of virus isolation success from clinical samples across a range of viral loads 27 inoculated in parallel on Vero E6 and human airway epithelia (HAE) showed lower success of 28 virus isolation in HAE, suggesting an overestimation of actual infectiousness in humans using 29 Vero E6 cell lines, commonly considered as reference. 30 31 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted May 19, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 

Understanding the window of Severe Acute Respiratory Syndrome coronavirus 2 (SARS- 2) infectiousness is essential for the control of the coronavirus disease 19 pandemic. Thanks to its high sensitivity and rapidity, real time RT-PCR remains the gold 35 standard for diagnosing SARS-CoV-2. However, detection of viral RNA by this method does not 36 necessarily equate with the presence of infectious viral particles, the requisite for 37 transmission. 38

Although resource-intensive (as dependent on biosafety level-3 facilities) and not suitable for 39 routine diagnostic purposes, inoculation of patient's specimens on cultured cells is to date the 40 only method to confirm presence of infectious SARS-CoV-2 in a sample. A number of studies 41 highlighted two determinants of the presence of infectious particles: SARS-CoV-2 RNA copy 42 numbers (RNAc) per mL in the original specimens, as determined by RT-PCR, and the symptom 43 duration. The probability of isolating viable virus hence appears to be drastically reduced 44 below 5.4-7 log10 RNAc/mL, and after more than one week of symptoms [1] [2] [3] . 45

The vast majority of these investigations used Vero E6 cells, derived from the kidney of an 46 African Green Monkey, although several other conventional Human cell lines, such as Caco2 47 (colorectal adenocarcinoma) and Calu3 (lung cancer cells) were also found to be susceptible 48 [4] . As they are interferon-production deficient [5] and express the virus receptor [6], Vero E6 49 cells are highly susceptible to SARS-CoV-2 and thus are the most widely used reference cell 50 line for isolation. However, these cells do not mimic the in vivo situation of the SARS-CoV-2 51 entry site, which is the human respiratory tract. The use of reconstituted human primary 52 airway epithelial cells (HAE) better reflects SARS-CoV-2 infection characteristics in vivo. 53

To assess of the threshold for the presence of infectious virus in this more relevant model 54 system, we compared virus isolation success from clinical samples across a range of viral loads 55 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted May 19, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 (VLs) inoculated in parallel on Vero E6 and HAE. We found that this threshold is higher in HAE 56 compared to Vero E6, suggesting the presence of infectious virus as determined by Vero E6 57 would not necessarily be sufficient to lead to an infection in HAE. 58 In this study, we used nasopharyngeal swabs collected in viral transport medium from 59 symptomatic adult individuals presenting at the outpatient testing center of the University 60 Hospitals of Geneva, spanning the first pandemic wave in spring 2020 and a second pandemic 61 wave in fall 2020, that tested positive for SARS-CoV-2 by RT-PCR (Cobas® SARS-CoV-2 Test, 62

Cobas 6800, Roche, Switzerland). All samples were collected from patients within the first 5 63 days post onset of symptoms (dpos), diagnosed between April and September 2020. All 64 viruses circulating during the investigated time period were characterized by the D614G 65 mutation, but no variants of concern were circulating in Switzerland during that time. VLs were 66 calculated for the E gene target as previously described [7] . In order to avoid loss of infectivity, 67 all samples were frozen at -80°C after diagnostic testing. Their inoculation was performed 68 immediately after a single thawing (there were no repeated freeze-thaw cycles). Regarding 69 virus isolation, 100 µl of the original clinical sample was inoculated in parallel on Vero E6 cells 70 grown in 48 well plates and in HAE (MucilAir TM commercially available, Epithelix SARL), with 71 an approximate number of cells for both culture systems of 2E+05 cells per well. Infections 72 were performed at 37°C under a 5% CO2 atmosphere as previously described [8, 9] . Viral 73 replication was assessed by quantitative RT-PCR from RNA extracted from the supernatant 74 collected at 1 hour post infection (hpi) for Vero E6 and from apical tissue washes 3hpi for HAE, 75 and at the end of the experiment 6 days post infection (dpi). Successful virus isolation was 76 determined by at least 3log10 folds increase of RNAc between baseline and 6 dpi. 77

In total, 38 primary clinical specimens were inoculated in parallel, with a VL ranging from 5.7-78 9.0 log10 SARS-CoV-2 RNAc/mL in the original sample. The overall frequency of successful virus 79 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted May 19, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 isolation was 27/38 in Vero E6 and 12/38 in HAE ( Figure 1A and Table S1 ). No growth in either 80 cell culture system was observed in samples below 5.8 log10 RNAc/mL. The lowest VL from 81 which infectious virus could be isolated in Vero E6 was 5.9 log10 RNAc/mL and 6.3 log10 82 RNAc/mL for HAE ( Figures 1A) . Although two samples with rather low viral load of 6.3 and 6.5 83 log10 RNAc/mL showed successful isolation in HAE, consistent isolation of virus was only 84 observed in samples with a VL of 7.7 log10 RNAc/mL and higher. Using Probit analysis, the 85 probability of a sample being infectious in Vero E6 and HAE was below 5% when VL was lower 86 than 5.5 and 6.5 log10 SARS-CoV-2 RNAc/mL, respectively (p value <0.05). No correlation was 87 found between the number of dpos within the first 5 dpos and successful viral growth in both 88 models (in order to increase the likelihood of infectious virus presence, only samples from 89 patients  5 dpos were selected) (Table S1) . 90

Upon successful isolation, the virus showed replication to higher viral loads in Vero E6 (mean 91 VL of 12.1 log10 SARS-CoV-2 RNAc/mL, SD ± 0.7, range 11.1-14.06) compared to HAE (mean VL 92 of 10.3 log10 SARS-CoV-2 RNAc/mL, SD ± 1.4 range 6.1-11.4) at the end of the experiment (6 93 dpi, Figure 1B ). In conclusion, upon comparative virus isolation, we could recapitulate findings 94 from earlier studies on Vero E6 with successful virus isolation in a similar range as previously 95

reported. In the HAE model, however, virus isolation was only consistently successful in 96 samples with a VLs of ≥7.7 RNAc/mL ( Figure 1A ), but there were two outliers of successful 97 isolation with a much lower VL. In conclusion, Vero E6 cells are shown to be more permissive 98 for SARS-CoV-2 isolation and yield virus isolates from samples with lower VLs than HAE. 99 Furthermore, upon successful isolation, SARS-CoV-2 replicates in Vero E6 to higher virus titers 100 than in HAE. 101

Lower success of virus isolation in HAE, and thus a higher amount of infectious virus particles 102 needed to start an infection, could hint towards an overestimation of actual infectiousness in 103 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted May 19, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 humans when only using Vero E6 cell lines as a reference. This could mean that the actual 104 transmission risk would be lower than findings based on Vero E6 culture data and that 105 transmission-relevant infectious virus shedding is shorter and/or lower than what was 106 previously estimated. Furthermore, antigen-based rapid diagnostic tests, which have a lower 107 sensitivity than PCR but may detect infections with VLs as low as 6 log10 RNAc/mL [10], would 108 thus be an even better at identifying infectious individuals than previously believed. As the infectious dose of SARS-CoV-2 still needs to be determined in vivo [12] , and HAEs do 114 not completely recapitulate the in vivo situation, our study cannot be used to change existing 115 guidelines on isolation or discharge criteria. It nevertheless emphasizes the effect of the cell 116 lines used to culture the virus and thus supports the use of models that more closely reflect 117 the in vivo situation, such as HAE in air-liquid interface culture rather than conventional cell 118 lines, in order to better understand transmission risks of SARS-CoV-2 in patients. 119

Acknowledgments 120

We thank Catia Alvarez for excellent technical support and Erik Boehm for English proof 121 reading (Geneva Centre for Emerging Viral Diseases, Geneva University Hospitals, Geneva, 122 Switzerland). 123

This work was supported by the Private HUG Foundation, by the Pictet Charitable Foundation 125 and by the Swiss National Science Foundation (grant Nr. 196644, 196383) . 126 127 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Table S1 . Sample characteristics 176 177 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 19, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 

Virological assessment of hospitalized 129 patients with COVID-2019

Predicting infectious SARS-CoV-2 from