key: cord-0779000-n41xkgfu
authors: Mallm, J.-P.; Bundschuh, C.; Kim, H.; Weidner, N.; Steiger, S.; Lander, I.; Börner, K.; Bauer, K.; Hübschmann, D.; Benes, V.; Rausch, T.; Trevisan Doimo de Azevedo, N.; Telzerow, A.; Jost, K. L.; Parthe, S.; Schnitzler, P.; Boutros, M.; Müller, B.; Bartenschlager, R.; Kräusslich, H.-G.; Rippe, K.
title: Local emergence and decline of a SARS-CoV-2 variant with mutations L452R and N501Y in the spike protein
date: 2021-04-29
journal: nan
DOI: 10.1101/2021.04.27.21254849
sha: ea9946b8001715cdac6d73289b477c0c9e95cc46
doc_id: 779000
cord_uid: n41xkgfu

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are replacing the initial wild-type strain, jeopardizing current efforts to contain the pandemic. Amino acid exchanges in the spike protein are of particular concern as they can render the virus more transmissible or reduce vaccine efficacy. Here, we conducted whole genome sequencing of SARS-CoV 2 positive samples from the Rhine-Neckar district in Germany during January-March 2021. We detected a total of 166 samples positive for a variant with a distinct mutational pattern in the spike gene comprising L18F, L452R, N501Y, A653V, H655Y, D796Y and G1219V with a later gain of A222V. This variant was designated A.27.RN according to its phylogenetic clade classification. It emerged in parallel with the B.1.1.7 variant, increased to >50% of all SARS-CoV-2 variants by week five. Subsequently it decreased to <10% of all variants by calendar week eight when B.1.1.7 had become the dominant strain. Antibodies induced by BNT162b2 vaccination neutralized A.27.RN but with a two-to-threefold reduced efficacy as compared to the wild-type and B.1.1.7 strains. These observations strongly argue for continuous and comprehensive monitoring of SARS CoV 2 evolution on a population level.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing Other RBD mutations that provide partial resistance to neutralizing antibodies include K417N/T in P.1 and B.1.351 (3, 4, 12) and L452R in B.1.427/1.429 and B.1.617 (5) (6) (7) (8) 19) . L452R is a marker mutation of the B.1.427/1.429 strain from California (5) (6) (7) and is found together with the E484Q mutation in a newly emerging B.1.617 strain from India (8, 20) . Outside the RBD, two other S mutations have been associated with a phenotype. The D614G mutation, that occurred early in the pandemic, has been associated with increased transmissibility (21, 22) , and the L18F exchange has been linked to faster spreading of SARS-CoV-2 in England (23) . VOC B.1.1.7 has become dominant in the UK in late 2020 accounting for >90% of all infections in 12/2020 and has spread to other regions of Europe and beyond, where it became dominant as well (24) . In Germany, B.1.1.7 accounted for the vast majority of all new infections in April 2021 (9) . Similarly, VOC P.1 has been responsible for a second wave of infections in Brazil with the epicenter in Manaus (4) and VOC B.1.351 is becoming the major cause of in South Africa (3) with both viruses also spreading globally (1) . A study of 2,172 samples collected in California reported that B.1.427/B.1.429 has emerged in late 2020 and increased to >50% of all infections by end of January 2021 with a calculated 18.6-24% increase in transmissibility relative to wild-type (6) . B.1.617, another strain with the L452R mutation, has been linked to surge in the number of cases in April 2021 during the second COVID-19 wave in India (20) . Viral genome sequencing is of crucial importance to track the emergence of mutations that define existing and new VOC. By applying a tiling amplicon scheme in combination with multiplex PCR, entire SARS CoV-2 genomes can be sequenced with sufficient coverage in a cost-effective manner (25) (26) (27) . The comprehensive sequence information obtained by this strategy provides direct insight into the evolution of SARS-CoV-2 genomes and the spreading of variants. In addition, marker mutations identified by sequencing can be exploited to design melting curve-based PCR screens for the rapid detection of relevant variants (28, 29) .

Here, we have conducted whole virus genome sequencing of SARS-CoV-2 positive samples from the Rhine-Neckar district in Germany during the first quarter of 2021. We report on the local emergence of a variant designated as A. 27 .RN that simultaneously carries the N501Y and L452R mutations in the S gene, and its subsequent displacement by variant B.1.1.7.

Variant A. 27 .RN was isolated from patient samples and characterized in tissue culture.

Antibody neutralization assays with sera from BNT162b2 vaccinees showed a moderate decrease in neutralizing titers for A. 27 .RN compared to SARS-CoV-2 wild-type, but better neutralization than for the B.1.351 variant. Our results provide evidence for the transient appearance of a variant of potential concern, carrying the signature N501Y mutation. These observations indicate important differences regarding population spread between N501Y strains that are not recapitulated in infectivity assays in cell culture. Furthermore, they underpin the importance of unbiased surveillance by whole genome sequencing of SARS-CoV-2 positive samples on a population level.

For SARS-CoV-2 PCR and sequencing analysis, RNA was isolated from nasopharyngeal and oropharyngeal swabs applying automated magnetic bead-based nucleic acid extraction protocols. Either the QIASymphony, DSP Virus/Pathogen mini Kit (Qiagen) or Chemagic, Viral DNA/RNA 300 Kit H96 (PerkinElmer) were used, following the manufacturers' protocols and using the respective devices. The sample input volume was 140 µl, spiked with 10 µl per sample of the internal control (TIB Molbiol) and the elution volume was set to 100 µl. To confirm the mutation pattern after viral passaging in tissue culture, RNA was extracted with the QIAamp Viral RNA body fluid kit which was carried out on the QIAcube system (Qiagen) with manual lysis according to the manufacturer's protocol. 

SARS-CoV-2 genomes were sequenced with two approaches, based on the Nextera library generation approach (Cov-seq) and the ARTIC protocol (25) 

England Biolabs (NEB, p/n E7658), respectively. For Cov-seq, cDNA was generated with SARS-Cov2 specific primers and a PCR handle in 384-well plates. Reverse transcription (RT) of 2 µl of purified RNA was conducted with the Maxima reverse transcriptase (Thermo Fisher Scientific) and 7.5% PEG-8000. After 50 min at 50 °C and RT inactivation, PCR was performed by adding KAPA PCR mix and custom forward primers and a single reverse primer complementary to the PCR handle. Due to overlapping primer design, the RT and PCR were performed in two separate reactions for each sample. cDNA was purified with SPRI beads at a 0.5x bead ratio and selected samples were measured with Qubit and Tapestation. cDNA . 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 April 29, 2021. ; https://doi.org/10.1101/2021.04.27.21254849 doi: medRxiv preprint was diluted to 0.3 to 0.5 ng/µl and libraries were prepared with the Illumina Nextera kit using a downscaled version with the mosquito system in 384-well plates. The libraries from 384 samples were individually indexed, pooled and purified with SPRI beads.

The ARTIC sequencing generally followed the recommended protocol, but we could further expedite and scale sample processing by implementation of three modifications: (i) With the help of NEB's technical support team it was possible to omit the magnetic bead clean-up step after PCR amplification of cDNA. (ii) The clean-up of adaptor ligated fragments was conducted on an automated liquid-handling system. (iii) After the final PCR, libraries were pooled, and bead purified with SPRI beads at a 0.9x ratio.

All libraries generated were sequenced in pools of up to 384 samples with the Illumina NextSeq 500/550 sequencing instrument in paired-end mode with a read-length of 75 bases from each direction.

Sequencing data from Cov-seq were processed using the nf-core viralrecon pipeline (30, 31) with the following settings: (i) NC_045512.2 genome, (ii) metagenomic protocol without primer sequence removal, (iii) no duplicate filtering, (iv) minimal coverage of 20 for variant calling, and (v) maximum allele frequency of 0.9 for filtering variant calls. Primer sequences were not removed due to the protocol's specific amplicon design. Variant calls and reconstructed consensus sequences from iVar were used (32) . For the data analysis of the ARTIC sequencing, paired-end sequencing data of read-length 75 bases were filtered for sequencing adapters (trim galore) and host-read contamination (33) . Remaining reads were aligned to the NC_045512.2 reference genome using bwa (34) . Alignments were sorted and indexed using samtools (35) and quality-controlled using alfred (36) . We then masked priming regions with iVar (32) . Variant calling employed FreeBayes (37) and bcftools (38) for quality-filtering and normalization of variants. Samtools, bcftools and iVar were also employed for viral consensus sequence generation. Lineage and clade classification of the SARS-CoV-2 sequences was carried out using Pangolin (39) and Nextclade as part of the Nextstrain framework (40) .

Vero E6 cells (ATCC#1586) were cultured in Dulbecco's modified Eagle medium (DMEM, Life Technologies) containing 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin and 1% non-essential amino acids (complete medium). The original SARS-CoV- 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 April 29, 2021. ; https://doi.org/10.1101/2021.04.27.21254849 doi: medRxiv preprint at 800xg for 10 min. Virus stocks were produced by one additional amplification in Vero E6 (passage 4), aliquoted and stored at -80 ⁰C. Virus titers were determined by plaque assay as reported earlier (41) . To isolate SARS-CoV-2 variants, nasopharyngeal swabs collected from PCR confirmed SARS-CoV-2-positive patients were resuspended and used to inoculate Vero E6 cells that were seeded in 24 well plates at a density of 2.5x10 5 cells per well. 100 µl of clinical specimen were used to inoculate cells in the first well and five-fold serial dilutions were added to cells in subsequent wells. After 1 h, inoculum was removed and replaced by fresh complete DMEM. Cells were inspected by microscopy for the appearance of a cytopathic effect on a daily basis. In most cases, extensive CPE was observed at four days post-inoculation.

Supernatant was collected (passage 0), centrifuged at 1,200 x g for 5 min and 0.35ml were used to inoculate 3x10 6 Vero E6 cells, seeded into a T25 cm 2 flask one day prior to inoculation, in a total volume of 5 ml complete DMEM. After two days, CPE was observed, supernatant was collected (passage 1) and used for SARS-CoV-2 genome sequencing. Virus stocks were produced by one further passage in Vero E6 cells (passage 2; B.1.1.7, B.1.351 and A27.RN), virus titer was determined, and stocks were stored at -80 ⁰C.

Sera from healthy donors vaccinated with the BTN162b2 SARS-CoV-2 mRNA vaccine were collected at day 10-28 after the second vaccine dose and used immediately or stored at 4°C until use. IgG reactivity against SARS-CoV-2 S1RBD was analyzed by a commercial chemoluminescence immunoassay (Siemens sCOVG; #11207377) run on a Siemens ADVIA Centaur XP instrument according to the manufacturer's instructions. Interference of sera with RBD-ACE2 interaction was assessed by a titration experiment using serial 1:3 dilutions of sera and a commercial ELISA based test system (GenScript SARS-CoV-2 Surrogate Virus Neutralization Test Kit; #L00847), following the manufacturer's instructions. Based on the results from these analyses, six sera covering a range of relative reactivities were selected for virus neutralization assays (Supplementary Table S3 ).

Neutralization titers were determined in titration experiments on Vero E6 cells as described previously (42, 43) . Vaccinee sera were serially diluted and mixed with the respective SARS-CoV-2 variants. After 1 h at 37 °C, the mixture was added to Vero E6 cells and 24 h later, cells were fixed in the plates with 5% formaldehyde. Virus replication was determined by immunostaining for the viral nucleoprotein using an in-cell ELISA. Values were normalized to the non-treated control (100% infection) and non-infected cells (0% infection). Dose response curves were generated by non-linear regression sigmoidal dose response analysis with Prism 7 (GraphPad Software).

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Vero E6 cells were seeded into 24 well plates at 2.5x10 5 cells per well. On the next day, cells were inoculated (MOI=0.05) for 1 h, inoculum was removed, and fresh medium was added. . 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 April 29, 2021 Subsequently, the additional S gene mutation A222V was found in samples collected in February and the relative fraction carrying this additional mutation increased to 10-30% in calendar week 7. The A222V mutation has been described as a marker mutation of the 20A.EU1 strain that has been spreading in Europe in 2020 (45) . The location of the mutated residues of A.27.RN in the S protein structure and in the RBD -ACE2 interface is depicted in Supplementary Fig. S1 . . 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.

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The sequences obtained were placed into the established SARS-CoV-2 phylogenetic tree (40) , yielding the results depicted in Fig. 1. A.27 is a newly designated lineage in clade 19B (46) .

Within this lineage, the A.27.RN sequences form a separate branch (Fig. 1) since they carry the unique D173G mutation in ORF3a, which was absent only in 10/166 cases (Supplementary Fig. 2B) . A.27 is clearly separated from the three currently prevailing N501Y containing strains (Fig. 1) . The latter, as well as the L452R carrying B. 

The first month of 2021 in the Rhine Neckar region was characterized by a rapid local spread of variant A.27.RN. During the period of calendar weeks 53/2020-11/2021 covered in our sample set, N501 wild-type variants were almost completely displaced by variants carrying the N501Y mutation ( Fig. 2A, Table S2 ). Initially, A.27.RN dominated and increased to a peak value of ~58% of all N501Y variants in calendar weeks four and five ( Fig. 2A, Table S2 ).

week seven with each of them comprising >25% of all samples sequenced (Fig. 2B) .

Subsequently the occurrence of A.27.RN decayed to a 10% fraction in calendar week eleven, while B.1.1.7 became dominant and was found in more than 85% of all samples at that time (Table S2) As a faster and less expensive screening readout, we employed Tm-PCR to detect S gene variants N501Y, K417N/K417T and the HV69/70 deletion with data shown here for calendar weeks 5-11 (Fig. 2C) . Samples were classified as potentially being B.1.1.7, B.1.351, P.1, N501Y only, HV69/70 deletion only or as unknown (Supplementary Fig. S3A) . Samples for which a valid result could not be obtained for at least one mutation were considered inconclusive. To assess the concordance of the "N501Y only" category with A. 27 Fig. S3B,C) . The remaining three samples (3%) were classified as P.3 (48) . The P.3 variant shares the B.1.1.28 ancestor with P.1 and carries E484K but has a distinguishing P681H mutation also present in B.1.1.7. P3 is . 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 April 29, 2021. ; https://doi.org/10.1101/2021.04.27.21254849 doi: medRxiv preprint indistinguishable from A.27 in the Tm-PCR scheme used here, since both variants have N501Y but lack HV69/70 deletion and K417N. Nevertheless, the "N501Y-only" category proved to be a good predictor of the A. 27 .RN variant in the tested cohort. By exploiting the information obtained from the sequencing data to define a limited subset of mutations, variants of interest can also be identified in a cost-efficient manner with faster turnaround times by the targeted Tm-PCR approach.

To assess replication and spread of A. 27 . 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 April 29, 2021. ; https://doi.org/10.1101/2021.04. 27.21254849 doi: medRxiv preprint This was reflected by a lower ratio of plaque-forming units to RNA copies for all three variants, arguing for lower specific infectivity of the variant virus particles as compared to the wild-type (Fig. 3A) . Furthermore, we noted a consistently reduced plaque size in the case of the B.1.1.7 variant compared to the other strains arguing for less efficient spread of B.1.1.7 in this tissue culture system (Fig. 3B) .

To evaluate the effect of S gene mutations found in A.27.RN on neutralization capacity of vaccine sera, we performed titration experiments with serum samples from BNT162b2 vaccine recipients. Six samples collected 10-28 days after the second vaccine dose that displayed a range of anti-S1 RBD IgG levels were chosen (Supplementary Table S3 (Fig. 4) . 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 April 29, 2021. ; https://doi.org/10.1101/2021.04.27.21254849 doi: medRxiv preprint Neutralizing titers of sera against the wild-type isolate varied from ~1:40 to 1:1,280, consistent with the different levels of IgG reactivity determined by ELISA. Accordingly, neutralization capacity against the A.27.RN varied somewhat between donors (Fig. 4A, B) but was notably reduced compared to wild-type in all cases. Averaged values of the relative difference in NT50 values between variants against wild-type virus yielded a fold-difference and 95% confidence interval (CI) of 2.6 (95% CI = 2.2 to 3.1) for A.27.RN, 0.9 (95% CI = 0.8 to 1.1) for B.1.1.7 and 4.3 (95% CI = 3.1 to 6.0) for B.1.351 (Fig. 4C) . These values were mostly in line with a recent study that also reports similar serum neutralization values for B.1.1.7 and wild-type but a somewhat higher factor of 9-12 for B.1.351 (12, 49) .

The mutational pattern of A.27.RN is different from other currently prevailing N501Y variants as it combines N501Y and L452R and carries additional S protein and other mutations.

According to public databases, the L452R mutation has been acquired by several other independent lineages across multiple countries and continents (5-7, 20, 46) . Such repeatedly emerging hot-spot mutations typically indicate strong and probably recent positive selection (17). The spreading profile of A.27.RN on a population level suggests that its transmissibility is higher than that of the SARS-CoV-2 wild-type strain but lower than that of the B. Our study revealed the regional and clustered appearance of a SARS-CoV-2 variant of potential concern, which rapidly became dominant locally but subsequently was outcompeted by another recently introduced variant on the population level. These results show that the presence of certain signature mutations is not sufficient to predict the spread of different SARS-CoV-2 variants. It can be speculated that the lack of the D614G mutation lowers the transmissibility of A27.RN, since D614G is found in all of the other variants discussed here ( Table 1 ) and has been associated with an increased SARS-CoV-2 fitness (21, 22) . Vaccine sera were capable of neutralizing A.27.RN, albeit with reduced neutralization titers. This effect was even more pronounced for B.1.351 in line with previous findings (12, 49) . Accordingly, it is expected that combinations of the phenotypically relevant L18F, K417N/T, L452R, E484R/Q, N501Y and D614G mutations in the S protein discussed here, as well as newly emerging ones, will become advantageous under immune selection. Continued and comprehensive surveillance of emerging SARS-CoV-2 strains and mutations by unbiased whole genome . 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. 

None declared. 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 April 29, 2021 . 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 April 29, 2021 . 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 April 29, 2021 . 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. Complete S protein after furin cleavage. (B) Complex of S protein RBD with ACE2 repressor visualization of S protein structure with mutated residues. N501 directly interacts with N330 of the receptor. In contrast, L452 is not in direct contact with ACE2 but creates as hydrophobic patch together with F490 and L492 on the RBD surface.

. 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 April 29, 2021 . 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 April 29, 2021 . 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 April 29, 2021. ; https://doi.org/10.1101/2021.04.27.21254849 doi: medRxiv preprint

The sequence of the A. 27 .RN variant as well as its A222V mutated from are provided in FASTA format as a zip file.

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The copyright holder for this preprint this version posted April 29, 2021. ; https://doi.org/10.1101/2021.04.27.21254849 doi: medRxiv preprint

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