key: cord-0902101-ham36q39 authors: Rosin, Nicole L.; Jaffer, Arzina; Sinha, Sarthak; Mulloy, Rory P.; Robinson, Carolyn; Labit, Elodie; Almeida, Luiz G.; Dufour, Antoine; Corcoran, Jennifer A.; Yipp, Bryan; Biernaskie, Jeff title: SARS-CoV-2 infection of circulating immune cells is not responsible for virus dissemination in severe COVID-19 patients date: 2021-01-19 journal: bioRxiv DOI: 10.1101/2021.01.19.427282 sha: a3e80fc17d5e946dbc35b7f4bb4658786c0e1145 doc_id: 902101 cord_uid: ham36q39 In late 2019 a novel coronavirus (SARS-CoV-2) emerged, and has since caused a global pandemic. Understanding the pathogenesis of COVID-19 disease is necessary to inform development of therapeutics, and management of infected patients. Using scRNAseq of blood drawn from SARS-CoV-2 patients, we asked whether SARS-CoV-2 may exploit immune cells as a ‘Trojan Horse’ to disseminate and access multiple organ systems. Our data suggests that circulating cells are not actively infected with SARS-CoV-2, and do not appear to be a source of viral dissemination. Coronaviruses are enveloped, non-segmented, positive-sense RNA viruses that can infect both 24 humans and many other mammals 3 . Of those that infect humans, some cause only mild 25 symptoms, such as hCoV-OC43 8 . This 'common cold' coronavirus shares only 56% genomic 26 sequence homology with SARS-CoV-2 (using BLAST search: 37% mortality rates respectively 9,10 and share 79% and 50% genomic sequence homology to 32 SARS-CoV-2, respectively 11 . containing macrophages in the bronchoalveolar lavage fluid (BALF) 22 . Furthermore, multiple 44 organ systems beyond the lung are also affected in severe COVID-19 disease 23 , and multiorgan 45 tropism has been reported 24 , which raises the possibility that visceral organ infection may be 46 Table 1 ). We detected one or more viral proteins in 57 the plasma of each of the five patients (Table 1 ). NSP3 (encoded by the ORF1ab gene) was 58 detected in all samples, while NSP3, the spike protein S1 and RNA-directed RNA polymerase 59 were found in three of the plasma samples. One sample, from patient UC_5, contained 5 SARS-60 CoV-2 specific proteins. 61 From whole blood, we isolated leukocytes and enriched for the lymphocyte population to ensure 64 that enough lymphocytes would be collected/assessed due to reported lymphocytopenia 26 . The 65 leukocytes and lymphocytes were mixed in equal ratios prior to conducting scRNAseq using the 66 suggests that aligning to our custom SARS-CoV-2 reference genome is a robust method of 94 detecting SARS-CoV-2, and taken together supports that peripheral immune cells do not contain 95 SARS-CoV-2 RNA. Of note, we examined one additional SARS-CoV-2 positive patient who 96 presented with a thrombotic stroke, but no respiratory symptoms or ARDS (and subsequently 97 was not included in the aggregated data presented here), who also lacked any detectable SARS-98 CoV-2 RNA in the isolated circulating immune cells. While anecdotal, given we studied only 99 one patient exhibiting clotting symptoms, it does suggest that this divergent clinical presentation 100 is also not due to direct infection of circulating platelets or other immune cells. Table 4) . 106 Unsurprisingly, the spike protein cell surface processing protease transmembrane protease serine 107 2 (TMPRSS2) and surface receptor (for host cell entry) angiotensin-converting enzyme 2 (ACE2 108 ) 29 were highly expressed in epithelial cells ( Figure 1 ). Macrophages expressed neuropilin-1 109 (NRP1), which has also been reported as a means of host cell entry (Figure 1) 30, 31 . Neutrophils do 110 not express ACE2, TMPRSS2 or NRP1, suggesting that they phagocytosed viral particles or 111 their products without being actively infected, and raises the possibility that macrophages may 112 do the same. 113 It is also important to note that the BALF samples were isolated using 10X Genomics 5' 115 technology, whereas the peripheral immune cells presented here, were isolated using 10X 116 Genomics 3' technology. Identification of SARS-CoV-2 RNA should theoretically be captured 117 using either technology. However, to ensure that coronavirus RNA could be detected in the 118 peripheral cells, we also prepared a control sample of hCoV-OC43 (a 'common cold' 119 coronavirus)-infected primary Human Umbilical Vein Endothelial Cells (HUVECs, Lonza) as a 120 control for 3' capture of viral RNA. OC43-infected HUVECs were processed identically to the 121 peripheral cells. We aligned the resulting sequences with a custom reference genome, again 122 based on GRCh38, with the hCoV-OC43 genome (NC_006213.1) appended, and were able to 123 reliably detect hCoV-OC43 RNA using our analysis pipeline ( FASTAs containing raw sequences were concatenated with the genome.fa file in GRCh38-3.0.0 211 reference. FASTA and GTF outputs following concatenation were provided as inputs to "fasta" 212 and "genes" arguments for cellranger mkref. Note that this approach did not generate a conjoined 213 genome containing both human and SARS-CoV-2 sequences as separate genomes. Instead this 214 approach added a new pseudogene called "SARS-CoV-2" to the human genome, as SARS-CoV-215 higher-depth GEMs to ensure each library received equivalent read count (Supplementary Table 219 2). with iodoacetamide solution (50mM) for 20min at room temperature. Samples were precipitated 257 by acetone/methanol, and 600µL ice-cold acetone was added followed by incubation at -20°C 258 overnight. A protein pellet was obtained by centrifugation (8,000 , 10min, 4°C) followed by 259 acetone drying (2min). Precipitated pellet was resuspended in100 µL of 50mM 260 triethylammonium bicarbonate (TEAB) buffer followed by tryptase digestion (5µg trypsin per 261 100µg of protein) overnight at 37°C. TMT-6plex™ Isobaric Labeling Reagents (90061, Thermo 262 Fisher) were resuspended in anhydrous acetonitrile and added to each sample (41µL TMT-263 6plex™ per 100µL sample) and incubated at room temperature for 1h. The TMT labeling 264 reaction was quenched by 2.5% hydroxylamine for 15min at room temperature. TMT labeled 265 samples were combined and acidified in 100% trifluoroacetic acid to pH < 3.0 and subjected to 266 C18 chromatography (Sep-Pak) according to manufacturer recommendations. Samples were 267 stored at -80°C before lyophilization, followed by resuspension in 1% formic acid before liquid 268 chromatography and tandem mass spectrometry analysis. Orbitrap first performed a full MS scan at a resolution of 120,000 FWHM to detect the precursor 282 ion having a m/z between 375 and 1,575 and a +2 to +4 charge. The Orbitrap AGC (Auto Gain 283 Control) and the maximum injection time were set at 4 x 10 5 and 50ms, respectively. The 284 Orbitrap was operated using the top speed mode with a 3 second cycle time for precursor 285 selection. The most intense precursor ions presenting a peptidic isotopic profile and having an 286 intensity threshold of at least 2 x 10 4 were isolated using the quadrupole (Isolation window (m/z) 287 COVID-19 viral RNA in circulation cells of 0.7) and fragmented using HCD (38% collision energy) in the ion routing multipole. The 288 fragment ions (MS2) were analyzed in the Orbitrap at a resolution of 15,000. The AGC and the 289 maximum injection time were set at 1 x 10 5 and 105ms, respectively. The first mass for the MS2 290 was set at 100 to acquire the TMT reporter ions. Dynamic exclusion was enabled for 45 seconds 291 to avoid of the acquisition of same precursor ion having a similar m/z (plus or minus 10ppm). Go to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE151969 302 To be released, please contact the authors to request early access. 303 To review GEO accession GSE156639 (HUVEC response to SARS-CoV-2 and OC43 gene 305 expression, 3 samples): 306 Go to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE156639 307 To be released, please contact the authors to request early access. 308 Table S1. Patient demographics Table S2. Single cell RNAseq sample data Table S3 . Average gene expression per cell for each sample Table S4 . 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Lymphopenia in severe coronavirus disease-2019 (COVID-19): systematic review and meta-analysis Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19 Comprehensive Integration of Single-Cell Data SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor Neuropilin-1 facilitates SARS-CoV-2 cell entry and provides a possible pathway into the central nervous system. bioRxiv Neuropilin-1 is a host factor for SARS-CoV-2 infection SARS-CoV-2 viral load is associated with increased disease severity and mortality Determination of 50% endpoint titer using a simple formula Integrating single-cell transcriptomic data across different conditions, technologies, and species All of the samples were aligned to a custom reference genome consisting of GRCh38-3.0.0 with MN908947.3 appended B) UMAP illustrating the detection of SARS-CoV-2 RNA (aligned to MN908947.3), and C) Violin plot illustrating detection of SARS-CoV-2 RNA A second dataset was generated from HUVECs infected with hCoV-OC43 at a titre of 3 TCID50/mL for either 12h or 24h. D) UMAP illustrating the detection of hCoV-OC43 RNA in aggregated samples, and E) Violin plot illustrating detection of hCoV-OC43 RNA according to length of infection NR -conceived of the study, processed samples for 10X genomics, analysed scRNAseq, wrote the manuscript AJ -pre-processed all scRNAseq data CR, RPM, JAC -provided hCov-OC43, and conducted HUVEC transductions, and provided critical virology expertise EL -processed samples for 10X genomics SS -constructed all custom reference genomes LGA, AD -conducted proteomics and analysis BY -enrolled patients, collected samples, cowrote the manuscript JB -supervised experiments, cowrote the manuscript The authors declare that they have no competing interests.