key: cord-0886032-c8tiioy8 authors: Wylezich, Claudia; Schaller, Tina; Claus, Rainer; Hirschbühl, Klaus; Märkl, Bruno; Kling, Elisabeth; Spring, Oliver; Höper, Dirk; Schlegel, Jürgen; Beer, Martin; Dintner, Sebastian title: Whole-genome analysis of SARS-CoV-2 samples indicate no tissue specific genetic adaptation of the virus in COVID-19 patients’ upper and lower respiratory tract date: 2021-08-20 journal: Diagn Microbiol Infect Dis DOI: 10.1016/j.diagmicrobio.2021.115520 sha: 0257010134316ddbfdbf553a074ea7a396a8e0af doc_id: 886032 cord_uid: c8tiioy8 Sample panels of SARS-CoV-2 cases were retrospectively whole-genome sequenced. In three individuals, samples of upper and lower respiratory tract resulted in identical sequences suggesting virus stability including the spike protein cleavage site. In a fourth case, low-level intra-host genomic evolution and a unique 5-nucleotide deletion was observed. While SARS-CoV mainly affects the lower respiratory tract tissue, SARS-CoV-2 replicates effectively in the nasopharynx leading to a more efficient transmission of the virus [1] . Irrespective of the severity of SARS-CoV-2 infection, the virus consistently replicates in the upper and lower respiratory epithelia [2] . Until now, sequence stability of SARS-CoV-2 in the upper and lower respiratory tract (URT and LRT, respectively) is insufficient explored. Therefore, sample panels comprising different clinical specimen of lethal SARS-CoV-2 cases were retrospectively investigated (Augsburg study [3] , Supplementary Table 1 ). After decease, postmortem analyses were performed between April 6 and May 13, 2020 (n=13, Figure 1A ); samples were taken from different sites of URT and LRT and were tested for SARS-CoV-2 RNA using RT-qPCR (Supplementary Table 2 ). Selected samples were subsequently used for highthroughput sequencing (HTS) to compare whole-genome sequences, which originated from different specimens per case (URT vs. LTR, Figure 1A) . HTS was performed with samples when more than one sample per case was available and/or the RT-qPCR cycle threshold values were ≤25. Methods are described in the Supplementary Methods. With this rationale, 13 whole-genome SARS-CoV-2 sequences were obtained. Four cases (AS-007, AS-011, AS-012, AS-015) were represented by two to four sequences obtained from different clinical samples. In two cases, all analyzed SARS-CoV-2 whole-genome sequences obtained from one individual were identical, in particular pharynx and/or trachea, bronchus, and pleura samples (cases marked in blue and red, Figure 1B Figure 1) , and go back to an original virus variant exhibiting an S-614D type that was circulating early in the pandemic until it was replaced by the more infectious S-614G type [5] . The sampling date of the green-marked case AS-007 (April 2020) matches with the main circulation period of this virus type. Furthermore, these S-614D type sequences found in pharyngeal and pleura samples showed a 5-nucleotide (nt) deletion in ORF8 ( Figure 1B) , which leads to a truncation of 15 amino acids when CTG can be used as an alternative start codon, and introduces a frameshift ( Figure 1C ). If no alternative start codon can be used, the protein would be completely lost. Such a 5-nt deletion of ORF8 was not described before, i.e., it is not existing in any SARS-CoV-2 sequence submitted to NCBI so far (until March 12, 2021). In addition to the deletion, we detected one transition (position 2492, Figure 1B ) in the SARS-CoV-2 sequence of the pharynx sample compared with the pleura sample. Single nucleotide variants (SNVs) were not detected in any of the analyzed samples except sample 4032 (pharynx, green-marked case AS-007). Here, we detected two SNVs, namely T9817C (frequency 24.4%) and C12775T (frequency 12.7%; 0.53 strand bias for both variants), that were both not found in the corresponding pleura sample. Importantly, none of the sequences showed any changes or SNVs in the SARS-CoV-2 spike protein cleavage site. Intra-host variability during the course of COVID-19 was investigated and compartmentalized virus replication was observed in former SARS-CoV-2 studies [6, 7] . However, neither significant differences regarding nucleotide diversity and SNVs nor a compartment-specific pattern were detected between samples from different respiratory compartments [6] . Also in our study, not investigating the evolutionary process during the Table 4 ). The soon decease might be due to the comorbidities, i.e., a malign lung tumor in progressed stage (poorly differentiated adenocarcinoma stage 4) besides atrial fibrillation, and aortic valve stenosis (Supplementary Table 5 ). However, we cannot fully exclude a longer persistence of the infection before clinical admission. With respect to the unique ORF8 deletion detected in this patient, different deletions within the SARS-CoV-2 ORF8 have been described before [9, 10, 11] . The accessory gene encoded by ORF8 is suspected to have an important role in immune response of the host [2, 9] . Despite the small patient cohort, our results do not indicate a general necessity of tissue specific adaptation but rather an accidental occurrence of SNVs; they also suggest stability of the spike protein cleavage site within different compartments of patient's respiratory tract. However, sequences of one individual showed a unique deletion of ORF8 and additionally some sequence 6 differences between the URT and LRT sequences. This might be a patient-induced effect due to poor health condition of the individual. In addition, it can only be speculated whether a longerterm replication of the virus could occur as in the case of other described ORF8 deletions. Overall, based on the present data with a limited sample size, this provides a hint and needs to be proven in further cohort studies. Furthermore, the study underlines the importance of wholegenome sequencing to detect variants and to reveal intra-host evolution of the virus. This study was approved by the local institutional review board (2020-18), and written informed consent was obtained from next of kin. No potential conflict of interest was reported by the authors. This work was financially supported by the German Federal Ministry of Food and Agriculture through the Federal Office for Agriculture and Food, project ZooSeq, grant number 2819114019. Virological assessment of hospitalized patients with COVID-2019 Spatial and temporal dynamics of SARS-CoV-2 in COVID-19 patients: A systematic review and meta-analysis Postmortem examination of patients with COVID-19 A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology Tracking changes in SARS-CoV-2 spike: evidence that D614G increases infectivity of the COVID-19 virus Compartmentalized replication of SARS-Cov-2 in upper vs. lower respiratory tract assessed by whole genome quasispecies analysis. Microorganisms Evolution of viral quasispecies during SARS-CoV-2 infection Case study: Prolonged infectious SARS-CoV-2 shedding from an asymptomatic immunocompromised individual with cancer Structure of SARS-CoV-2 ORF8, a rapidly evolving immune evasion protein Evolutionary dynamics of the SARS-CoV-2 ORF8 accessory gene Discovery and genomic characterization of a 382-nucleotide deletion in ORF7b and ORF8 during the early evolution of SARS-CoV-2. mBio KH and RC performed the autopsies, BM, OS, KH collected clinical data. SD, EK performed the primary swab analyses by PCR, CW sequenced genomes and analyzed data. CW, BM, DH, MB wrote the paper. All authors conducted the study, collected data, read and corrected the manuscript and approved the final version Rainer Claus We thank Patrick Zitzow and Maria Eberle for excellent technical support, and Arbor Biosciences for providing SARS-CoV-2 baits for sequencing. 7 Supplementary data to this article can be found online at {https://doi.org/…}.