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-2 related coronaviruses (SARS2r-CoVs) prior to the COVID-19 pandemic. Here, we examine the novel SARS2r-CoV we previously identified in game animal metatranscriptomic datasets sequenced by the Nanjing Agricultural University in 2022, and find that sections of the partial genome phylogenetically group with Guangxi 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, it is also found in 10 additional NGS datasets from 5 separate mammalian species and is likely related to contamination by a laboratory researched virus. Absence of bat mitochondrial sequences from the datasets, the fragmentary nature of the virus sequence and the presence of a partial sequence of a cloning vector attached to a SARS2r-CoV read suggests that it has been cloned. We find that NGS datasets containing the novel SARS2r-CoV are contaminated with significant Homo sapiens genetic material, and numerous viruses not associated with the host animals sampled. We further identify the dominant human haplogroup of the contaminating H. sapiens genetic material to be F1c1a1, which is of East Asian provenance. The association of this novel SARS2r-CoV with both bat CoV and the GX PCoV clades is an important step towards identifying the origin of the GX PCoVs.

Identification of a novel HKU4-related coronavirus in single-cell datasets and clade viral host analysis

“…This indicates that the infection of pangolins by HKU4-related CoVs is extremely rare. This is consistent with our assessment that pangolins are unlikely to be natural reservoirs or intermediate reservoirs for SARS-related CoVs (Jones et al, 2021;2022a;2022b) or HKU4-related CoVs.…”

“…This is over two years after SARS-CoV-2 related CoVs were identified in pangolin RNA-Seq datasets (Liu et al, 2019;Wong et al, 2020;Lam et al, 2020), despite extensive pangolin sampling and sequencing (Lee et al, 2020;Hu et al, 2020;Li et al, 2020;He et al, 2022). Given the cross-contamination of the single cell RNA-Seq datasets in BioProject PRJNA747757 identified here, along with concerns for previous pangolin coronavirus sequencing datasets raised by multiple authors (Hassanin, 2020;Chan and Zhan, 2020;Zhang, 2020;Jones et al, 2021Jones et al, , 2022aJones et al, , 2022b we reviewed the RNA-Seq datasets containing these three recently published pangolin-hosted HKU4-related CoVs.…”

“…It is interesting to note two other recent betacoronavirus discoveries made through forensic analysis of contamination of NGS datasets also exhibit basal sister relationships to closely related CoVs. GX_ZC45r-CoV exhibits a basal sister phylogenetic relationship to SARS-2-related Guangxi pangolin CoVs in the NSP4, NSP10 and partial RdRp regions (Jones et al, 2022b). While HKU4-HZAU-2020, a HKU4-related cDNA clone, again sits in a basal sister relationship to its three most closely related HKU4-related CoVs (Jones et al, 2023).…”

Discovery of a novel merbecovirus DNA clone contaminating agricultural rice sequencing datasets from Wuhan, China

“…Indeed, Katz et al (2021) identified that in early 2020, over 2,000 Public Health England surveillance bacterial next-generation sequencing SRA datasets were likely contaminated with SARS-CoV-2 sequences. In some cases, the identification and characterization of cross-contaminating reads may provide useful biosecurity and or research insights (Lewis et al, 2020; Csabai and Stéger 2021; Quay et al, 2021; Jones et al, 2022).…”