Forensic analysis of novel SARS2r-CoV identified in game animal datasets in China shows evolutionary relationship to Pangolin GX CoV clade and apparent genetic experimentation

Abstract

Pangolins are the only animals other than bats proposed to have been infected with SARS-CoV-2related coronaviruses (SARS2r-CoVs) prior to the COVID-19 pandemic. Here we examine thenovel SARS2r-CoV we previously identified in game animal metatranscriptomic datasetssequenced by He et al. (2022) and find that sections of the partial genome phylogenetically groupwith Guangxi (GX) pangolin CoVs (GX PCoVs), while the full RdRp sequence groups with bat-SL-CoVZC45. While the novel SARS2r-CoV is found in 6 pangolin datasets, the same CoV isalso found in 10 additional NGS datasets from 5 separate mammalian species and is likelyrelated to contamination by a laboratory researched virus. Absence of bat mitochondrialsequences from the datasets, the fragmentary nature of the virus sequence and the presence of apartial sequence of a cloning vector attached to a SARS2r-CoV read suggests that it has beencloned. We find that NGS datasets containing the novel SARS2r-CoV are contaminated withsignificant Homo sapiens genetic material, and numerous viruses not associated with the hostanimals sampled. We further identify the dominant human haplogroup of the contaminatingH.sapiens genetic material to be F1c1a1, which is of East Asian provenance. The association ofthis novel CoV with both bat CoV and the Guangxi pangolin CoV (GX PCoV) clades is animportant step towards identifying the origin of the GX PCoVs.

Forensic Analysis of Novel SARS2r-CoV Identified in Game Animal Datasets in China Shows Evolutionary Relationship to Pangolin GX CoV Clade and Apparent Genetic Experimentation

“…It is of further concern that out of nine published GX PCoVs, only one unfiltered/non highly enriched pangolin tissue SRA dataset has been provided to support assembly of a GX PCoV, GX_P3B, a partial genome with 86% coverage of GX_P2V [24,25]. The dataset is of low quality with read lengths highly skewed to very short lengths, and is contaminated with SARS-CoV-2 reads.…”

“…Consequently, it has been proposed that SARS-CoV-2 acquired its RBD via recombination with, or from an ancestor in common with a GD PCoV [23]. However, the SARS2r-CoV in the Liu et al datasets may be contamination related rather than pangolin hosted given the low number of SARS2r-CoV reads, presence of human genomic sequences, presence of non-pangolin hosted virus sequences in similar abundance as SARS2r-CoV sequences, and correlation of of SARS2r-CoV sequences with high bacterial content [24,25].…”

“…For phylogenetic analysis the following workflow was used: 100 SARSr-CoV genomes with highest Blastn percentage identity to each of the assembled NSP10 + RdRp region, NSP4 region and bat-SL-CoVZC45 were de-duplicated. The GX_WIV [28] genome sequence was added to the set. The sequences were then aligned using the MUSCLE algorithm in UGENE with default settings.…”

“…GX_ZC45r-CoV exhibits a basal sister relationship to GX CoVs with unanimous support. GX_WIV [28] exhibits a more divergent sequence in this region than related GX CoVs (Figure 6). A maximum likelihood tree implemented using PhyML using a GTR+G+I model shows the same basal sister relationship of GX_ZC45r-CoV to GX PCoVs (Supplementary Figure S24a).…”

“…GX PCoVs were first reported by Lam et al [26] in February 2020 from analysis of frozen tissue samples collected in Guangxi province between 2017 and 2018. The GX PCoVs form a separate clade to GD PCoVs and are more distantly related to SARS-CoV-2 [24,[26][27][28]. The spike proteins of GX PCoVs have a higher amino acid similarity in the S1 N-Terminal Domain (NTD) to SARS-CoV-2 than GD PCoVs, but conversely a lower similarity in the RBD to SARS-CoV-2 and GD PCoVs (Supplementary Figures S1 and S2).…”