Genome determinants of furin cleavage in various European SARS-related bat coronaviruses

Collection and processing of samples

Bat(R. euryale, R. blasii, R. Ferro Equinum When R. Hipposideros) Sampled in 4 countries (Bulgaria, Italy, Slovenia, Spain)^{15,16 16,18 18}.. All animals were treated in accordance with national and European law on the protection of animals (EU Council Directive 86/609 / EEC). No animals were sacrificed during this study. All animal handling and sampling was done by trained personnel, and animal safety and comfort were top priorities for minimally invasive sampling (fecal collection). The captured bats were immediately released from the net and placed in a cotton bag for 2-15 minutes to allow them to settle before inspection. Bats were placed in bags to generate fecal pellets and transferred to 500 µl RNAlater RNA stabilizing solution (Qiagen, Hilden, Germany) for sample processing. Licenses for sampling bats using mist nets, hand nets, or harp traps have been obtained from their respective countries and authorities. Bulgarian Ministry of the Environment, License No. 192 / 26.03.200913, Ministry of the Environment, Italy, License No. 192 / 26.03.200952, Slovenia Environment Agency, License No. 35701-80 / 200453, Spain, Xunta de Galicia Rural Counseling Biodiversity Conservation Service for, authorization number 52 / 2010ns13697.

Analysis of samples by reverse transcription PCR

Specimens were screened for the presence of coronavirus RNA using nested reverse transcription PCR (RT-PCR). RNA-dependent RNA polymerase (RdRp) gene. Briefly, RT-PCR is a QIAGEN (Hilden, Germany) one-step RT containing 5 μl RNA, 200 nM primer PC2S2 (isomolar mixture of TTATGGGTTGGGATTATC and TGATGGGATGGGACTATC), 900 nM primer PC2As1 (isomolar mixture). -Performed using a PCR kit. TCATCACTCAGAATCATCA, TCATCAGAAAGAATCATCA, and TCGTCGGACAAGATCATCA) and 1 μl QIAGEN one-step RT-PCR kit enzyme mix. The amplification protocol consisted of 30 minutes at 50 ° C. 15 minutes at 95 ° C; 10 cycles of 20 seconds at 94 ° C, 30 seconds with a 1 ° C decrease per cycle starting at 62 ° C, 40 seconds at 72 ° C. 30 cycles of 20 seconds at 95 ° C, 30 seconds at 52 ° C, 40 seconds at 72 ° C³⁵.. For further phylogenetic analysis, these amplicons were extended to> 800 bp fragments towards the 5’end using a 5’primer sequence: 5′-CTTCTTCTTTGCTCAGGATGGCAATGCTGC-3′; SP3195, 5′- ATACTTTGATTGTTACGATGGTGGCTG-3′; SP3374, 5′-CTATAACTCAAATGAATCTTAAGTATGC-3′; GrISP1, 5′-TTCTTTGCACAGAAGGGTGATGC-3′; and GrISP2, 5′-CTTTGCACAAAAAGGTGATGCWGC-3^{17 17}..1 bat SrC The spiked glycoprotein gene (called BB99-04; GenBank Acc No. KR559017) was fully characterized using a nested RT-PCR primer set (supplementary data). 2).

8 bat S1 / S2 genomic region (346 bp; SARS-CoV-2 Wuhan strain GenBank Acc. No. MT019529, position 23,422-23,767) SrC (GenBank Acc No. KC633198, KC633201, KC633203-205, KC633209, KC633212 and KC633217) were characterized using a heminestized RT-PCR assay using the following oligonucleotides: panSARS-S1S2-F1 (TDGCTGTTGTHTAYCARGATGT). ), PanSARS-S1S2-F2 (CAGATGTWAAYTGYACWGATGT) and panSARS-S1S2-R (CAGATGTACATDKTACAATCBAC). The extended S2 region for phylogenetic analysis (691 bp; SARS-CoV-2 Wuhan strain 23,422-24,112) was amplified using the same forward primers, but panSARS-S1S2-R2 (AGDCCATTRAACTTYTGHGCACA) reverse. I used a primer. Briefly, RNA is reverse transcribed at 50 ° C for 30 minutes using the SSIII One Step Kit (Thermo Fisher), then PCR at 94 ° C for 15 seconds, 58 ° C for 20 or 45 seconds, 72 ° PCR. The cycle was repeated 45 times. 1 minute at C. The second PCR was performed under the same conditions as the first round without reverse transcription. The PCR amplicon was sequenced by Sanger.

Follow the manufacturer’s instructions for a PCR amplicon of the S1 / S2 genomic region (346 bp fragment) and a MiSeq system with the MiSeq reagent kit v2 (500 cycles) to detect single nucleotide polymorphisms within the S1 / S2 site. Used to sequence. Sequence reads from the library were mapped to the corresponding S1 / S2 genomic sequences in Geneious 9.1.8. Custom script using Python module pysam (https://github.com/pysam-developers/pysam) Was used to determine the nucleotide distribution of a particular genomic position from a read alignment.

Phylogenetic analysis

Tblastx search for the full spike sequence in Bulgarian SrC (GenBank Acc No. GU190215) Classification ID 11118 (Coronaviridae) Implemented on December 28, 2021, excluding taxonomy ID 2697049 (SARS-CoV-2). Hits with a percentage of identity less than 80% are non-SrC It was not included in the dataset because it is a sequence. SARS-CoV sequences from infections or clones, and sequences <27,000 nt, or sequences with gaps in the region encoding spikes were excluded, resulting in 88 sequences. One reference sequence for each SARS-CoV (NC004718) and SARS-CoV-2 (MT019529) and nine sequences from the newly generated European bats in this study have been added to the final dataset of 99 sequences. It was created.Coronavirus frequently recombines³⁶ Only the S2 region (495 nt) of the 691 nucleotide fragment was used for phylogenetic analysis.Maximum likelihood phylogeny was generated using FastTree^{37 37} Version 2.1.10 with GTR replacement model and 1000 bootstrap duplications.Local support values ​​are based on the Shimohira Hasegawa (SH) test³⁸..

In silico analysis

Secondary structure was modeled using the UNAfold web server^{39 39}.. Furin cleavage site was predicted using ProP v.1.0b ProPeptide CleavageSitePrediction software.^Ten.. The sequences were taken from the NCBI Taxonomy website and downloaded all the sequences of the species. Duvinaco virus (HCoV-229E), Melbecovirus (MERS-CoV), Setracovirus (HCoV-NL63) and Embecovirus (HCoV-HKU1 and HCoV-OC43). Duplications, incomplete genomes, sequences from infections, and clones were excluded from all datasets. At this stage of the analysis, only the NCBI reference sequence of the human genome was left in the dataset. For Sarbecoviruses, the tBlastx search dataset is SrC-Systematic occurrence in the supplementary figure. 1 was used by. Translation adjustments were made in Generic 9.1.8. The accession numbers for all viruses tested with ProP are listed in the supplemental data. 3..Translated 816nt amino acid identity RdRp Fragment calculated with MEGA11⁴⁰ Use the pairwise delete option.

Data acquisition of HCoV sequences to investigate conservation of FCS in human sequences

The dataset used for ProP FCS prediction contained only one human reference sequence. However, the preservation of FCS in human-derived sequences was investigated as follows. Blastp search for partial spike sequences adjacent to S1 / S2 sites (SARS-CoV, NC004718, position (position) 23,139–24,191; HCoV-229E, NC002645, position 21,923–22,954; HCoV-NL63, NC005831, position 22,329–23,414; HCoV-OC43, NC006213, pos. 25,524–26,675; HCoV-HKU1, NC006577, pos. 24,823–25,977 and MERS-CoV, NC019843, pos. 23,358–24,449) nr Database as of January 3, 2022. To limit the results to human-derived sequences only, the blast search was performed with classification IDs 111137 (HCoV-229E), 290028 (HCoV-HKU1), 1335626 (MERS-CoV), 277944 (HCoV-NL63) and 31331 (HCoV-). OC43). For SARS-CoV, seed Coronavirus associated with severe acute respiratory syndrome (Category ID 694009) is selected and non-human viruses are excluded according to the classification ID (1283333, 2790772, 1283332, 442736, 349342, 349343, 347537, 347536, 349344, 1508227, 1415834, 1415851, 1415852, 1699360, 1699361). rice field. 285945, 698398, 1478703, 1503296, 1503299, 1503300, 1503301, 1503302, 1503303, 2042697, 2042698, 2697049). As a result of the Blastp search, 157, 60, 378, 102, 348, 94 hits were generated for HCoV-229E, HCoV-HKU1, MERS-CoV, HCoV-NL63, HCoV-OC43, and SARS-CoV, respectively. The complete record was downloaded and manually curated in Geneious 9.1.8. However, with the exception of infections, clones, genetically modified sequences, and sequences that partially covered the reference sequence, a final dataset containing 104, 58, 180, 55, 264, and 40 proteins was obtained. Sequences of HCoV-229E, HCoV-HKU1, MERS-CoV, HCoV-NL63, HCoV-OC43, SARS-CoV, respectively. In Geneious 9.1.8, the protein sequences were aligned using: Maft^{41 41} It uses an automatic algorithm and the BLOSUM62 scoring matrix. The FCS save was manually inspected.

Due to the huge amount of SARS-CoV-2 available sequences, we downloaded the metadata for all sequences available in GISAID to CET on January 11, 2022 at 10:55 am. Only sequence metadata that meets GISAID’s complete and high coverage criteria was used to create the final dataset containing metadata for 4,824,313 sequence entries. A custom Python script was used to extract the GISAID sample ID of a sequence that was completely or partially deleted within the SARS-CoV-2 FCS motif (amino acid position / motif). ₆₈₂RRAR₆₈₅) Or sequences with at least one amino acid substitution at position R682 or R685, all of which are likely to result in non-functional FCS. All of these criteria were consistent with 304 SARS-CoV-2 sequences of human origin. The sequence was downloaded from the GISAID database and aligned with the Wuhan reference strain MT019529 of Geneious 9.1.8. Maft..

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