key: cord-0779001-x4f31nnq authors: Sonnleitner, S. T.; Prelog, M.; Sonnleitner, S.; Hinterbichler, E.; Halbfurter, H.; Kopecky, D.; Almanzar, G.; Koblmueller, S.; Sturmbauer, C.; Feist, L.; Horres, R.; Posch, W.; Walder, G. title: The mutational steps of SARS-CoV-2 to become like Omicron within seven months: the story of immune escape in an immunocompromised patient. date: 2022-03-07 journal: nan DOI: 10.1101/2022.03.04.22271540 sha: f1ca740fa680e61c9a30d3f028a2b16e63d7789c doc_id: 779001 cord_uid: x4f31nnq We studied a unique case of prolonged viral shedding in an immunocompromised patient that generated a series of SARS-CoV-2 immune escape mutations over a period of seven months. During the persisting SARS-CoV-2 infection seventeen non-synonymous mutations were observed, thirteen (13/17; 76.5%) of which occurred in the genomic region coding for spike. Fifteen (15/17; 88.2%) of these mutations have already been described in the context of variants of concern and include the prominent immune escape mutations S:E484K, S:D950N, S:P681H, S:N501Y, S:del(9), N:S235F and S:H655Y. Fifty percent of all mutations acquired by the investigated strain (11/22) are found in similar form in the Omicron variant of concern. The study shows the chronology of the evolution of intra-host mutations, which can be seen as the straight mutational response of the virus to specific antibodies and should therefore be given special attention in the rating of immune escape mutations of SARS-CoV-2. In December 2019 the Wuhan Municipial Health Commission (China) reported a cluster of 3 5 cases of pneumonia of unknown etiology to the WHO China Country Office. By the beginning 3 6 of 2020 it was confirmed that a novel coronavirus later named severe acute respiratory 3 7 syndrome coronavirus (SARS-CoV-2), was the causative agent (1) . SARS-CoV-2 spreads 3 8 easily and effectively among human beings with a basic reproduction number (R0) of > 2 (2, 3 9 3). Following this rapid human-to-human transmission and intercontinental spread the WHO 4 0 declared a global pandemic in March of 2020. The first cases in Austria were reported in 4 1 Ischgl, Tyrol, as early as February 2020 -and East Tyrol was considered one of the first 4 2 hotspot areas in Central Europe. 4 3 While mutations are common in RNA viruses and mostly will not make a significant 4 4 difference, some mutations proved to provide SARS-CoV-2 with a selective advantage, such 4 5 as increased transmissibility or increased escape from specific antibodies (4) (5) (6) (7) (8) . Those 4 6 …number of mutations that are found in comparable expression in a variants of concern 2 1 2 (VOC) or a variant of interest (VOI). 2 1 3 Table 3 : Overview of A) substitutions and B) deletions in the SARS-CoV-2 genome over a 2 1 4 seven-months study period in an immunocompromised patient. substitutions and deletions, i.e. polymorphic substitutions and deletions.. Column 2 shows 2 1 7 substitutions and deletions in comparison to the reference genome Wuhan (GenBank: 2 1 8 MN908947.3, RefSeq: NC_045512.2), strain-specific, manifested since the beginning of the 2 1 9 infection and maintained throughout the seven-months study period. Column 3 is a 2 2 0 chronology of all mutations acquired by SARS-CoV-2 during the intra-host evolutionary 2 2 1 process. * … mutations of concern or mutations which are described in the context of 2 2 2 immune escape. Column 4 shows temporary mutational events which occurred once and did 2 2 3 not occur any more in the following sequence. Underlined mutation sites are also found in the Omicron variant, but with a different 2 2 7 substitution. immunocompromised patient. All acquired and preserved genetic adaptations occurred in the 2 3 3 regions ORF1a (n = 1), ORF1b (n = 1) and spike (n = 9). 2 3 4 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) Chronology of acquired mutations 2 3 6 In the underlying clinical case the substitutions emerged in the following chronological order : 2 3 7 S:Y144-emerged immediately after the increase of the specific antibody titre at day 117 as a 2 3 8 temporary mutation, followed by E484Q (day 129), which could not assert itself against 2 3 9 E484K and was displaced at least 7 days later (day 136). Furthermore, we found the 2 4 0 substitutions S:N354K (day 158, 164, 171 und 182), S:R346I (day 164) and ORF1a:T3284I 2 4 1 (day 171), S:D950N (day 171) as well as the prominent S:P681H on day 182 (Fig. 2) . Three 2 4 2 of the six acquired substitutions (50%) have already been described as typical mutations 2 4 3 acquired by diverse variants of concern. 2 4 4 Thirteen of the seventeen acquired substitutions (76.5%) occurred in the genomic region 2 4 5 coding for spike, and one each in the regions coding for ORF1a:T3284, ORF3a:V255X (day 2 4 6 73), ORF8:Y73C* (day 73) and N:S235F* (day 136) (Fig. 2) . Other mutations appeared temporarily and were subsequently replaced by the wildtype Six temporary mutations have already been described previously, all of them prominent 2 5 2 variations known in the context of variants of concern (https://covariants.org/shared-2 S:del(9) described in the Beta variant, N:S235F, known as a typical substitution of the Alpha 2 5 8 variant and S:H655Y, described in the context of the Beta as well as the Omicron variants. All these temporarily recurring mutational events did not establish permanently but 2 6 0 disappeared again or were dominated again by the wildtype variant. Figure 3 shows the 2 6 1 acquired und temporarily acquired mutations of strain EPI_ISL_2106199 in the region coding 2 6 2 for spike and demonstrates the high concordance of the acquired adaptations with described 2 6 3 variants of concern, above all the Alpha and the Omicron variant (15/17; 88.2%). course of the prolonged infectious phase are already described mutations in variants of 2 6 8 concern. Ten of the 17 spike mutations occur in a similar or identical way in the Omicron strain EPI_ISL_21061 convergently evolved in other variants of concern, mainly in the Alpha 2 7 7 and the Omicron variant. the region coding for spike. Overall, 17 persistent or temporary spike mutations were 2 8 3 evolved, whereas 9 (52.9%) turned out to be temporary and were subsequently replaced by 2 8 4 the wild-type variant. * … temporary mutations; S1 … spike 1; S2 … spike 2; hr … heptad 2 8 5 repeat; RBD … receptor binding domain; The mutations marked in orange are also found in 2 8 6 the Omicron variant (B.1.1.529) in similar or identical expression (10 out of 17), mutations 2 8 7 marked in red are found in other variants of concern (3 of 17; 17.6%). Thirteen of the 17 2 8 8 mutations (76.5%) acquired in the course of the prolonged infectious phase are already 2 8 9 described mutations in variants of concern. 2 9 0 2 9 1 Overall, SARS-CoV-2 developed eleven persistent mutations during the study period of 140 2 9 2 days as well as eleven temporary mutational events. The chronology of intra-host mutational 2 9 3 events is displayed in Figure 4 . patient with adequate humoral and lacking cellular immune response. The study period 2 9 8 comprised 140 days of almost permanent viral shedding. High-quality next-generation 2 9 9 sequences could be obtained at 14 time-points during the seven-month study period (starting 3 0 0 on day 73, ending on day 207 with the last SARS-CoV-2 positive swab) and disclosed the 3 0 1 chronological development of mutational events of SARS-CoV-2 as an answer to a unilateral 3 0 2 immune response with strong antibody answer but lack of specific T-cells. In the first swab sample, whole genome sequencing did not detect any spike mutations in the 3 0 5 investigated strain compared to the reference genome. First spike variants appeared as 3 0 6 E484K on day 133 as a heterozygotic mutation in 41.3% of the targeted reads. On day 136 3 0 7 the proportion of E484K increased to 76% and, after more than seven days (day 143) the 3 0 8 new variant dominated with 100%, but decreased to 76.8% again on day 158. On day 171 3 0 9 the spike variant P681H was observed for the first time with a proportion of 24% and 3 1 0 dominated within a couple of weeks reaching 100% on day 182. Three of the six acquired substitutions (50%) are previously described substitutions of 3 1 2 immune escape variants, namely: S:E484K, S:D950N and S:P681H. red line graph shows the mean frequency of all mutations at a given day. The occurrence of 3 2 0 mutations did not occur straight-line, but in a fluctuating course, with frequent replacement by 3 2 1 the wildtype variant. The fluctuating occurrence of adaptive mutations 3 2 4 The emergence of adaptive mutations did not occur in a linear but fluctuating fashion. Frequently, new mutations arose at a certain time point to be later replaced by the wildtype 3 2 6 variant, again. As shown in Figure 5 , the mutation rate shows an oscillating course with 3 2 7 . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 7, 2022. ; https://doi.org/10.1101/2022.03.04.22271540 doi: medRxiv preprint peaks around day 125, increasing until day 182. Simultaneously, the viral load decreased 3 2 8 continuously until the patient had several consecutive negative SARS-CoV-2 qPCR tests 3 2 9 since day 232 and is therefore considered to be cured from COVID-19 . 3 3 0 3 3 1 Intra-host evolutionary history 3 3 2 The intra-host evolution of the strain EPI_ISL_2106199 from day 73 and the quasi-species 3 3 3 arising from it in the course of the intra-host evolution form a distinct clade in the consensus 3 3 4 tree and group together. The clade is embedded in a random composition of complete 3 3 5 Austrian strains and variants of concern found in Europe and described and uploaded to 3 3 6 GISAID platform in the same study period from January to May 2021 (Figure 6) . Early 3 3 7 Austrian sequences of the Omicron variant from December 2021 were included 3 3 8 subsequently. in prominent variants of concern and typical Austrian strains sequenced in the same 3 4 5 investigation period, downloaded from GISAID and completed by early Austrian sequences In this unique case report we described the dynamics of intra-host mutational events in an 3 5 2 immunocompromised patient during a seven-months period of prolonged viral shedding and 3 5 3 proven infectivity. We considered the possible influence of a quantitatively strong but 3 5 4 regarding binding capacities probably functionally ineffective humoral antibody response and 3 5 5 a lacking cellular immune response on the site-directed mutagenesis of SARS-CoV-2. Our sequencing approach resulted in high-confidence variant identification and robust 3 5 7 genome-wide coverage and enabled the establishment of a chronology of immune escape 3 5 8 mutations. In addition, previously undescribed site-directed base-exchanges, found in the 3 5 9 regions ORF1a and b (n = 2;ORF1a:T3284I, ORF1b:L714-), ORF3a (ORF3a:V255X) and spike due to the relatively low neutralizing ability of the detected IgG which is also supported by the 3 6 4 low avidity of the specific IgG and impaired avidity maturation over time. Administration of 3 6 5 IVIG was not able to enhance the clearance of SARS-CoV-2. Cellular immunity was 3 6 6 diminished in this patient and the lack of adapted T cell-mediated immune defence may have 3 6 7 contributed to the inefficient clearance. The substitution rate for SARS-CoV-2 was estimated as (8-9) x 10 -4 nucleotides per site per 3 6 9 year (32). This is comparable to previously reported substitution rates of SARS-CoV (8.0-3 7 0 23.8 * 10 -4 ) (33) and MERS-CoV (11.2 * 10 -4 ) (34, 35) and comparable to the reported 3 7 1 substitution rates for Influenza A (4-5 * 10 -3 ) and Influenza B (2 x 10 -3 ) virus in the 3 7 2 haemagglutinine gene (36) . From this substitution rate it can be estimated that SARS-CoV-2 undergoes about one genetic change every other week (32). In comparison, the nucleotide 3 7 4 substitution rate per site and per year for Ebola (EBOV Makona) is estimated to be ~1.2 * 10 -3 7 5 . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 7, 2022. ; https://doi.org/10.1101/2022.03.04.22271540 doi: medRxiv preprint 3 (37) and for HIV-1 (3.21-4.06) * 10 -3 (38) . Interestingly, the evolutionary rate stayed 3 7 6 constant throughout the first months of infection but decreased slightly after the increase of 3 7 7 specific antibodies on day 124. We did not find elevated intra-host substitution rates 3 7 8 compared to the general rate reported for SARS-CoV-2 (32). This unaltered intra-host 3 7 9 evolutionary rate compared to the global average evolutionary rate suggests that these 3 8 0 mutations in an immunocompromised patient, driven by specific antibodies, do not lead to 3 8 1 more frequent random genomic changes, but on the contrary to very specific targeted ones. 3 8 2 We assume that the presence of specific antibodies forced directional selection on retaining 3 8 3 or regaining infectiousness and thereby strongly favoured directional mutations at particular 3 8 4 sites, acting as immune escape mutations. compromising vaccines effectiveness (4, 6, (40) (41) (42) (43) (44) (45) (46) (47) . E484K is a well-established distinction of 3 8 8 the variants of concern (VOC) B. could also be detected as a temporary substitution. A further genomic change in the region 3 9 5 coding for spike was identified on position S:N354K on day 158 and had never been 3 9 6 described before. R346I was detected in the sequence of day 164. This mutation was 3 9 7 previously described as a reaction of SARS-CoV-2 after monoclonal antibody treatment, 3 9 8 seeming to maintain ACE2 binding activity (50) and has also developed in the VOI Mu, 21H, 3 9 9 B. The substitution P681H was observed for the first time in the sequence of day 171, whereby 4 0 5 the amino acid histidine (H) first appeared as a polymorphism to become the dominant and 4 0 6 finally fixed variant in the course of the next ten days. This transformation from proline (P) to 4 0 7 histidine is relatively well studied and implicates a modification in the neighbouring furin 4 0 8 cleavage site at the junction of the spike protein receptor-binding (S1) and fusion (S2) 4 0 9 domains (51). Nine permanent mutations were found in the spike-coding region. More precisely, four are Of the eleven acquired adaptive mutations, only two were found outside the spike-coding 4 2 2 regions, namely L714-in ORF1b (day 158) and T3284I in ORF1a (day 171). ORF1a and 4 2 3 ORF1b are coding regions for non-structural proteins (nsp) (52) . ORF1a:T3284I is located in 4 2 4 the region encoding for nsp5. Nsp5 is regarded as the main protease, cleaves viral 4 2 5 . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 7, 2022. It is remarkable that nine of the eleven persistent mutations (81.8%) acquired in the course of 4 3 0 the prolonged infection had previously been described in the context of immune escape and 4 3 1 were assigned to diverse variants of concern. Our bioinformatic analyses revealed that 75% 4 3 2 of the novel mutations in our investigated strain also occur in variants of concern, whereas 4 3 3 the highest concordance was found between strain EPI_ISL_2106199 and the Omicron 4 3 4 variant (50%). Furthermore, we found dynamic mutational events with fluctuations between 4 3 5 the wildtype and the variational mutation. Nine of these temporary mutations (9 of 11; 81.8%) 4 3 6 have also been described in the context of variants of concern (Center for Disease Control 4 3 7 and Prevention; https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-info.html). In the 4 3 8 close proximity of the acquired deletion in ORF1b:L714-, which manifested homozygously, 4 3 9 additional conspicuous polymorphic sequences in amino acid position ORF1b:708-716 were 4 4 0 found. We measured frequent changes in substitutions and deletions. This certainly left us 4 4 1 with the impression of mutational escape manoeuvres. Also, this hotspot in directional 4 4 2 mutations encodes for nsp13, the helicase. We thus suggest that the accumulated mutations are results of an increased selection 4 4 4 pressure on spike, the key to entering the host cell. At the same time a second process takes 4 4 5 place intra-host, which exerts increased pressure and enforces continual reconstructions in 4 4 6 nsp13. The findings of these temporary mutations, which almost exclusively occurred in the 4 4 7 spike region, also fit this pattern very well. We managed to isolate SARS-CoV-2 from swabs at different time points, which is further 4 4 9 evidence for the continuous viability of the virus over the study period, given the evolutionary 4 5 0 dynamics of the different sequences. The isolation success correlated negatively with the Ct 4 5 1 value, a fact that has already been observed in previous studies (22) . Treatment with Rituximab resulting in depletion of particularly memory and effector B cells by 4 5 3 targeting CD20 is known to cause impaired antibody responses (58) (59) (60) . As naïve B cell 4 5 4 clones are less sensitive to Rituximab treatment due to their lower expression of CD20, a 4 5 5 robust immune response can also be assumed for those patients. Immunosuppressive number of immune escape mutations in a single patient, which to a strikingly high degree 4 6 5 evolved in parallel in various variants of concern. The chronology of mutation events during 4 6 6 seven months of infection shows a rapid accumulation of non-synonymous mutations which 4 6 7 in part were persistent, in part temporary or even repeatedly acquired and lost. 4 6 8 In summary, our case report documents the medical phenomenon of persisting SARS-CoV-2 4 6 9 infection in an immunocompromised patient with impaired humoral and cellular immune 4 7 0 response. Potential interference of specific antibodies led to a significant reduction in the viral 4 7 1 load, but at the same time generated sophisticated escape mechanisms while the cell- insufficient humoral immune defence. This led to the formation of highly specific virus 4 7 5 variants, highlighting the regions exposed to the highest intra-host selective pressure. Based 4 7 6 . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 7, 2022. ; https://doi.org/10.1101/2022.03.04.22271540 doi: medRxiv preprint on this observation one may hypothesize that immunocompromised patients pose a 4 7 7 particular risk to establish a source of immune escape mutants of SARS-CoV-2. Our study 4 7 8 also underlines the importance to protect these patients from SARS-CoV-2 infection by 4 7 9 modified vaccination strategies as well as to reinforce vaccination efforts to increase herd 4 8 0 immunity in general. Most importantly, the study points out the convergent evolution of 4 8 1 specific mutations in SARS-CoV-2, both in VOCs, VOIs and intra-host in the strain we 4 8 2 studied (EPI_ISL_2106199). Those specific, convergently evolving mutations reveal those 4 8 3 neuralgic positions in the SARS-CoV-2 genome that on the one hand represent its highest 4 8 4 fitness advantage, but on the other hand also uncovers its highest vulnerability and should 4 8 5 be considered as the probably most important points of attack in future vaccine and 4 8 6 therapeutics development. Germany) and an in-house enzyme-linked neutralization assay (ELNA) (14) at day 102, 124, 4 9 4 182 and 205 after the first positive PCR. The LIAISON® SARS-CoV-2 TrimericS IgG is a CLIA (Chemiluminescent Immunoassay) 4 9 6 which detects IgG antibodies reactive with the spike protein (S1/S2 domain). The assay was 4 9 7 performed on the LIAISON® XL Analyzer according to the manufacturer's instructions and 4 9 8 gives the arbitrary units per ml (AU/mL) according to the WHO International Standards for 4 9 9 the Anti-SARS-CoV-2-immunoglobulin-binding activity (NIBSC 20-136). 5 0 0 Microarray immunoblots 5 0 1 The ViraChip® assay detects temporal antibody profiles of different immunoglobulin classes 5 0 2 against S1, S2, and nucleocapsid (N) as well as against N of the four nonSARS human 5 0 3 coronaviruses in a commercial, miniaturized 96 wells protein microarray. The ViraChip® 5 0 4 assay is a useful tool to identify the epitope-specificity of IgG and IgA in serum samples. The 5 0 5 quantitative antibody measurement was performed on a ViraChip® Scanner using ViraChip® 5 0 6 Software. 5 0 7 Neutralization test 5 0 8 Neutralization ability of antibodies was determined performing an in-house enzyme-linked 5 0 9 neutralization assay (ELNA) as described elsewhere (14). Anti-IgG-SARS ELISA 5 1 1 Serum IgG antibodies against SARS-CoV-2 were determined by Serion agile SARS-CoV-2 ELISA with a sensitivity of 96.2% and a specificity of 100% according to manufacturer´s 5 1 3 instructions (Virion/Serion, Wuerzburg, Germany). Antibody activities above 15U/mL were 5 1 4 considered positive. 5 1 5 Anti-IgG-SARS-Avidity 5 1 6 Relative avidity index (RAI) was determined by a modification of the Serion agile SARS-CoV-5 1 7 2 IgG-SARS ELISA using 1M ammonium thiocyanate (NH 4 SCN) as a chaotropic agent as 5 1 8 described previously (15) (16) (17) . RAI values were considered as: RAI > 60% high avidity, 40% < 5 1 9 RAI < 60% as moderate, and RAI < 40% as low avidity in reference to other viral infections 5 2 0 (18). SARS-CoV-2 specific T cell response 5 2 2 . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 7, 2022. ; https://doi.org/10.1101/2022.03.04.22271540 doi: medRxiv preprint The ELISpot assay was performed using a commercially available precoated human SARS-5 2 3 CoV-2-specific IFN-γ ELISPOT kit according to the manufacturer´s protocol (AutoImmun 5 2 4 Diagnostika, GmbH, Germany; Cat.no. ELSP 5500). Peripheral blood was collected into 5 2 5 tubes coated with lithium-heparin (Vacuette, Greiner bio-one, Austria). PBMCs were 5 2 6 separated from plasma and whole blood by gradient density (FicoLite® -H, Linaris, 5 2 7 Germany). After washing with phosphate-buffered saline (PBS), depleting erythrocytes 5 2 8 (RBD-Lyse Buffer Life Technologies, 1xRBC Lysis Buffer 200ml; Invitrogen eBioscience, 5 2 9 USA REF: 00-4333-57) and washing again with PBS, cells were counted and resuspended in 5 3 0 x-vivo medium (X-VIVO TM-10 Serum-free hematopoietic cell medium; BEBP02-055Q, 5 3 1 Lonza, Switzerland). Briefly, a total of 2 x 10 5 PBMCs were incubated in duplicate with x-vivo as a negative 5 3 3 control, pokeweed mitogen (AutoImmun Diagnostika GmbH, Germany) as a positive 5 3 4 control,15-20mer peptide pools for SARS-CoV-2 (AutoImmun Diagnostika GmbH, Germany) 5 3 5 and PanCorona (AutoImmun Diagnostika GmbH, Germany) for the four nonSARS human 5 3 6 coronaviruses 229E, HKU1, NL63 and OC43 as a control of possible cellular cross-reactive 5 3 7 responses. After incubation at 37° C for 20 hours in a sterile and humidified atmosphere, plates were washed with washing buffer (AutoImmun Diagnostika GmbH, Germany) and 5 3 9 stained with the kit-specific reagents according to the manufacturer´s protocol. Plates were 5 4 0 then washed several times under running water and dried overnight. Spot forming units 5 4 1 (SFU)/100.000 cells were counted using an automated AID ELISPOT reader system 5 4 2 (AutoImmun Diagnostika GmbH, Germany). The assessment criteria for the ELISpots were a minimum of 50 SFU in the positive control 5 4 4 and a maximum of 10 SFU in the negative control according to the manufacturer´s definitions 5 4 5 (19, 20) . When those criteria were fulfilled, the stimulation index (SI) was calculated by 5 4 6 dividing the mean SFU numbers in the antigen-specific wells with the mean SFU numbers of 5 4 7 the negative control. The test was assessed negative with an SI < 2 according to previous 5 4 8 determination of the cut-off by well-defined pre-pandemic PBMC samples and by PBMCs 5 4 9 from SARS-CoV-2-naive individuals. The test was suggested to be poorly reactive with an SI 5 5 0 between 2 and 7 and reactive with an SI ≥ 7 as defined by the manufacturer (19) . According 5 5 1 to standardized laboratory procedures, in each assay, a standard laboratory control sample 5 5 2 of a high-reactive and a non-reactive PBMC sample, respectively, was run to determine inter-5 5 3 assay-variations. Only assays with less than two standard deviations of the high-reactive and 5 5 4 the non-reactive PBMC control sample, respectively, were defined valid. 5 5 5 Sample collection 5 5 6 Nasopharyngeal swabs were taken in a standardized way in home quarantine in the context 5 5 7 of primary care by a medical co-worker. KingFisher deep well plates (Thermo Fisher Scientific, Waltham, Massachusetts, USA) 5 6 7 containing 280µl isopropanol and 2µL Mag-Bind particles per well, using a KingFisher TM Flex 5 6 8 purification system (Cat. No. 5400620). 5 6 9 rtPCR 5 7 0 qPCR extracts were tested for SARS-CoV-2 by qRT-PCR using the Bio-Rad CFX96 system 5 7 1 (Bio-Rad, Germany) with a LightMix Modular Assay kit in accordance with the modified 5 7 2 . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 7, 2022. ; https://doi.org/10.1101/2022.03.04.22271540 doi: medRxiv preprint Charité guidelines (Corman et al., 2020) . 10µL of extracted RNA were added into 15µL 4x 5 7 3 Reliance One-Step Multiplex Supermix (Bio-Rad, Germany). Each 15µL mastermix 5 7 4 contained 12.5µL buffer solution, 0.25µl enzyme mix, 1.75µL of nuclease-free water and 5 7 5 0.5µL primer probe wHCoV (E-Gene, as well as N-Gene and Rdrp-Gene for confirmation). 5 7 6 Reactions were incubated at 55° C for 5 min and 95 C for 5 min in order to conduct reverse 5 7 7 transcription of viral RNA, sample denaturation and enzyme activation. These steps were 5 7 8 followed by PCR-amplification including 45 cycles at 95° C for 5 s, 60° C for 15 s and 72° C 5 7 9 for 15 s. Cooling was implemented at 40° C for 30 s. Results were interpreted based on the 5 8 0 Second Derivative Maximum (SDM) method. Positive results were confirmed by Rdrp and N-5 8 1 gene (21), samples with an initial Ct value lower than or equal to 37 were assigned to 5 8 2 repeated testing including extraction. A Ct value higher than 40 was considered negative. 5 8 3 Quantification of the viral load in the swabs was calculated via size standards of 1, 10, 100 5 8 4 and 1000 plaque-forming units (PFU)/mL. Standardization of viral stocks was carried out by 5 8 5 virus titration. Isolation was performed on VeroB4 cells as described elsewhere (22) . Virus titration 5 8 7 Confluent VeroB4 cells were cultured in Medium199 including 5% FCS in T75 tissue culture 5 8 8 flasks (Sarstedt, Germany) and transferred into 96-well tissue culture plates (Sarstedt, 5 8 9 Germany). Passage 1 isolates of SARS-CoV-2 were thawed from −80° C freezer and titrated 5 9 0 from 1:10 to 1:10 −12 in U-shaped 96-well plates (Greiner, Germany) and pipetted into each 5 9 1 corresponding well of the 96-well tissue culture plate. Plates were incubated at 36° C. Three 5 9 2 days post infection, incubation was stopped by gently removing the supernatant, washing the 5 9 3 cells three times with PBS and fixing cells in 1:1 ice-cold acetone-methanol. For easier 5 9 4 optical evaluation, cells were dyed by crystal violet staining and tissue culture infectious dose 5 9 5 of 70% (TCID 70 ) and PFU were calculated (Ramakrishnan, 2016). 5 9 6 Whole genome sequencing and mutational analysis 5 9 7 Libraries were prepared according to the Ion AmpliSeq™ SARS-CoV-2 Research Panel 5 9 8 (Thermofisher, USA), library construction and sequencing protocol with the Library Kit Plus 5 9 9 (Thermo Fisher Scientific, Waltham, Massachusetts, USA; Cat. No. 4488990). The 6 0 0 Amplicons were cleaned up with AMPure XP beads (Beckman Coulter, Germany) with a 1:1 6 0 1 ratio. The libraries were quantified using the Ion Library TaqMan TM Quantitation Kit (Cat. No . 6 0 2 4468802), normalizing, pooling and sequencing was performed using an Ion Torrent TM S5 6 0 3 Plus. Ion Torrent Suite software (v 5.12.2) of the Ion S5 sequencer was used to map the 6 0 4 generated reads to a SARS-CoV-2 reference genome (Wuhan-Hu-1; GenBank accession 6 0 5 numbers NC_045512 and MN908947.3), using TMAP software included in the Torrent Suite. 6 0 6 The following plugins were used: Coverage Analysis (v5.10.0.3), Variant Caller (v.5.12.04) 6 0 7 for mutation calls both with "Generic S5/S5XL (510/520/530) Somatic Low Stringency" 6 0 8 and "Generic S5/S5XL (510/520/530) Germ Line Low Stringency" default parameters and 6 0 9 COVID19AnnotateSnpEff (v.1.0.), a plugin specifically developed for SARS CoV 2 that can 6 1 0 predict the effect of a base substitution. No ultra-deep sequencing was performed and only 6 1 1 mutations visible in the stated analysis methods were listed and rated. 6 1 2 FASTA files containing the raw reads were inspected for quality criteria (mapped, targeted, 6 1 3 filtered reads, mean depth and uniformity) using Thermofisher Software. Multiple sequence 6 1 4 alignments were performed using Unipro UGENE (23) as well as MEGA X (Kumar et al., Hu-1. Viral genome assembly and screening for distinct mutations was performed online 6 1 7 using nextstrain.org (https://github.com/nextstrain/ncov/blob/master/defaults/clades.tsv; 6 1 8 https://clades.nextstrain.org/). The identification of pangolin lineages was carried out using 6 1 9 Pangolin software, v.2.4.2. (https://pangolin.cog-uk.io/). The generated full-genome 6 2 0 sequences are available at GISAID EpiCoV (https://gisaid.org/no.EPI_ISL_2106191-6 2 1 21061201). Additional sequences of frequent Austrian strains and prominent variants of 6 2 2 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 7, 2022. 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ModelFinder: fast model selection for accurate phylogenetic estimates concern, sequenced in the same study period, were retrieved from the GISAID EpiCoV 6 2 3 database (24) to calculate a consensus tree. 6 2 4Indels were coded using 'simple indel coding' (25) as implemented in 2matrix v.1.0 (70).The best-fit model of nucleotide substitutions (TIM2+F+I) was selected under the Akaike and 6 2 6 the Bayesian formation criteria using ModelFinder (71) as implemented in the PhyloSuite 6 2 7Software package (26) . Phylogenomic inference was based on a Maximum Likelihood (ML). 6 2 8An ML tree with 5000 ultrafast bootstrap replicates was inferred in the IQ Tree plugin (27, 28) 6 2 9of PhyloSuite.The frequency of the various mutations and the homology to the most widespread variants of 6 3 2concern (Alpha-, Beta, Gamma-and Delta-variant) were investigated based on BAM files.Reads from CoV-Seq samples were demultiplexed by using in-house tools. Reads 6 3 4originating from human were filtered out by mapping against hg38 with bwa-mem 0.7.17 (29). 6 3 5All reads not mapping to human were trimmed for adapters und quality by using Cutadapt 6 3 63.2 (30) . The trimmed reads were mapped with bwa-mem 0.7.17 to the SARS-CoV-2 6 3 7 reference MN908947.3 from the NCBI. Mutations were called using breseq 0.35.5 (31). 6 3 8Graphics were created using pandas 1.2 for Python 3. significance level of p < 0.05 was used for determining statistical significance. After testing 6 4 3for distribution (Kolmogorov-Smirnov-test), non-parametric continuous independent variables 6 4 4were compared using Mann-Whitney-U test for each time point. Dependent non-parametric 6 4 5variables were compared using Wilcoxon-rank test. The authors have no conflict of interest to declare. The generated full-genome sequences are available at GISAID EpiCoV (https://gisaid.org/no.EPI_ISL_2106191-21061201).