Anthropogenic Transmission of SARS-CoV-2 from Humans to Lions, Singapore, 2021

In Singapore, 10 captive lions tested positive for SARS-CoV-2 by real-time PCR. Genomic analyses of nanopore sequencing confirmed human-to-animal transmission of the SARS-CoV-2 Delta variant. Viral genomes from the lions and zookeeper shared a unique spike protein substitution, S:A1016V. Widespread SARS-CoV-2 transmission among humans can increase the likelihood of anthroponosis.


Multiplex PCR and Oxford Nanopore Sequencing and Assembly of SARS-CoV-2 Genomes
Two RNA samples from nasal swabs of two Asiatic lions (Cq = 23.05 and 24.47) and one individual fecal sample from an African lion (Cq = 36.02)were sequenced on the MinION Nanopore platform.One of the nasal swab samples (viral inactivated at 56 degrees for 2 hours) was re-extracted using QIAamp Viral RNA extraction kit following manufacturer's instructions.
All post-PCR, reverse transcription, and library purification steps were done with KAPA pure beads (Roche Sequencing).
Nasal swab RNA was converted directly to double-stranded complementary DNA (cDNA) by first-strand synthesis with the SuperScript III kit (Thermo Fisher Scientific) containing random hexamers.Briefly, 1 μL each of dNTPs and random hexamers (50 μmol, Invitrogen) were added to 11 μL of template RNA and incubated at 65°C for 5 minutes before placing on ice for 1 minute.Next, 1 μL of 0.1M DTT, 4 μL of 5′ First Strand buffer, 1 μL of RNaseOUT (Invitrogen) and 1 μL of SuperScript III were added to each cDNA conversion reaction assay of the previous step.The final 20 μL mix was incubated at 42°C for 50 minutes, followed by 70°C for 10 minutes, and placed on ice for 5 minutes.The PCR reaction products were purified, and cDNA was stored at −20°C until library preparation.
Briefly, first-strand cDNA was synthesized in a 20 μL reaction consisting of 7 μL of viral RNA from fecal samples, 50 pmol of 10 μmol primer A (5′-GTTTCCCACTGGAGGATA-NNNNNNNNN-3′), SuperScript III Reverse transcription (Thermo Fisher Scientific) and dNTPs (10 μmol), with incubation at 25°C for 5 minutes, followed by 50°C for 60 minutes and 70°C for 15 minutes.Second strand synthesis continued in the 20 μL cDNA mix forming a total volume of 24 μL with 2.5 μL Klenow buffer, 10 pmol Primer A, and dNTPs (1 μmol), with one-hour incubation at 37°C.The doublestranded cDNA was purified, and sequence-independent PCR amplification was done in a final reaction volume of 50 μL containing 2× LongAmp® Taq Master Mix (NEB), 50 pmol of 10 μmol primer B (5′-GTTTCCCACTGGAGGATA-3′).The PCR cycling was performed as follows: 94°C for 30 s, followed by 30 cycles of 94°C for 15 s, 50°C for 30 s and 65°C for 5 minutes, with a final extension at 65°C for 10 minutes, followed by purification of PCR products.
Multiplex PCR was used to enrich viral cDNA of all three samples using Q5 Hot-start HF polymerase with ARCTIC-CoV V1 and V3 protocols (J.R. Tyson et al., unpub.data, https://doi.org/10.1101/2020.09.04.283077),where the tiling primer schemes targeted SARS-CoV-2 genome-wide amplicons of 400-bp in length.PCR thermal cycling profiles consisted of 98°C at 30 seconds, followed by 25 (Cq <25 nasal swab samples) or 35 cycles (Cq: 36.02fecal samples) of 98°C for 15 seconds and 65°C for 5 minutes, before a final placement on ice for 5 minutes.Forward and reverse primer PCR reactions (odd and even primer pools) were conducted separately and pooled immediately after PCR, purified, and end-repaired with the NEB Ultra II companion kit.
MinION nanopore libraries were prepared using the Native Barcoding (EXP-NBD104) and ligation sequencing (SQK-LSK109) kits (Oxford Nanopore Technologies, Oxford, UK).Long reads are blasted to candidate reference sequences (NT&AA), aligned with aga (a combined NT&AA aligner, https://www.genomedetective.com/app/aga) to the top candidates, the consensus sequences are then constructed.Due to aligning in the NT and AA domain simultaneously, aga is able to detect and correctly handle frame shifts, and also corrects errors in monomer repeat regions as those errors are typical for nanopore sequencers.

Stitching of Sequences
The consensus contigs obtained from Genome Detective were aligned to the Singapore reference SARS-CoV2 sequence (EPI_ISL_6600690).Based on this reference, the gaps were determined.Passed nanopore reads (Q-score >8) were mapped using Minimap2 to this reference and the consensus bases (Base Quality >10) in the reads that encompassed these gaps were used to stitch the contigs together.The consensus cut off was at 80%, but when the cut off cannot be achieved, an N base was used as a fill in.No explicit depth cut off filter was applied in this analysis.Instead, the depth filter is implicitly defined with a consensus 80% cut off as demonstrated in Appendix 1 Table 1.

Finalized
cDNA libraries of 8-12 ng were loaded on three Flo-MIN106 R9.4 flow cells and sequenced for 22-36 hours.Phylogenomic Analyses MAFFT v. 7.490 to generate multiple sequence alignments for the 39 sequences intended for tree inference (Figure in main article text).The final alignment only consisted of coding regions and stop codons were verified to be present only at the terminal end of each gene.Next, RaxML-ng v1.1.0was used to infer a maximum-likelihood tree, using the GTR + I + G model of nucleotide substitution with 2000 bootstrap replicates, and the Wuhan-Hu-1 reference sequence (GenBank: NC_045512.2/ GISAID: EPI_ISL_402119) was used as the outgroup.Convergence of trees were attained at 1950 bootstraps.Inferred trees were visualized and annotated on Interactive Tree of Life v5 (iTOL) (4).Bioinformatic AnalysesGenome Detective Assembly of Nanopore Reads.Consensus sequences were generated by Genome Detective using a consensus algorithm that is reference based for 3GS reads (J.R. Tyson et al., unpub.data, https://doi.org/10.1101/2020.09.04.283077).For nasal swab samples from AS-M1 and AS-F1, the consensus viral sequences consisted of 29,822 bp, had mean read depths of 364.99 and 1,050.59,median read depths of 199 and 466, read depth ranges of 2-3,013 and 1-11,918, and mean base quality scores of 19.1 and 19.3.