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

HKU4-related coronaviruses (CoVs) are merbecoviruses related to Middle Eastern Respiratory Syndrome coronavirus (MERS-CoV). In 2022 and 2023, two HKU4-related CoV strains were discovered in Manis javanica (Malayan pangolin) metagenomic datasets derived from organ samples: HKU4-P251T and MjHKU4r-CoV-1. Together with the Tylonycteris robustula bat CoV 162275, which was discovered in 2022, pangolin CoVs HKU4-P251T and MjHKU4r-CoV-1 form a novel phylogenetic clade distinct from all previously documented HKU4-related CoVs. In this study, we identified a novel HKU4-related CoV in a pangolin single-cell sequencing dataset generated by BGI-Shenzhen in Shenzhen, Guangdong, China in 2020. The CoV phylogenetically belongs to the same newly identified clade. The single cell datasets were reported as generated from organ samples of a single pangolin that died of natural causes. 98% of the HKU4-related CoV reads were found in only one of the seven single cell datasets -- a large intestine cell dataset, cells of which exhibit low expression of DPP4. Bacterial contamination was found to be moderately correlated with HKU4-related CoV presence. We further identified with high confidence that the RNA-Seq dataset supporting one of four near identical variants of MjHKU4r-CoV-1 is a Sus scrofa (wild pig) metagenomic dataset, with only a trace level of Manis javanica genomic content. The presence of HKU4-related CoV reads in the dataset are almost certainly laboratory research-related and not from a premortal pangolin or pig infection. Our findings raise concerns about the provenance of the novel HKU4-related CoV we identify here, MjHKU4r-CoV-1 and its four near-identical variants.

Discovery of a novel merbecovirus cDNA clone contaminating agricultural rice sequencing datasets from Wuhan, China (preprint)

HKU4-related coronaviruses are a group of betacoronaviruses belonging to the same merbecovirus subgenus as Middle Eastern Respiratory Syndrome coronavirus (MERS-CoV), which causes severe respiratory illness in humans with a mortality rate of over 30%. The high genetic similarity between HKU4-related coronaviruses and MERS-CoV makes them an attractive subject of research for modeling potential zoonotic spillover scenarios. In this study, we identify a novel coronavirus contaminating agricultural rice RNA sequencing datasets from Wuhan, China. The datasets were generated by the Huazhong Agricultural University in early 2020. We were able to assemble the complete viral genome sequence, which revealed that it is a novel HKU4-related merbecovirus. The assembled genome is 98.38% identical to the closest known full genome sequence, Tylonycteris pachypus bat isolate BtTp-GX2012. Using in silico modeling, we identified that the novel HKU4-related coronavirus spike protein likely binds to human dipeptidyl peptidase 4 (DPP4), the receptor used by MERS-CoV. We further identified that the novel HKU4-related coronavirus genome has been inserted into a bacterial artificial chromosome in a format consistent with previously published coronavirus infectious clones. Additionally, we have found a near complete read coverage of the spike gene of the MERS-CoV reference strain HCoV-EMC/2012, and identify the likely presence of a HKU4-related-MERS chimera in the datasets. Our findings contribute to the knowledge of HKU4-related coronaviruses and document the use of a previously unpublished HKU4 reverse genetics system in apparent MERS-CoV related gain-of-function research. Our study also emphasizes the importance of improved biosafety protocols in sequencing centers and coronavirus research facilities.

Statistical challenges for inferring multiple SARS-CoV-2 spillovers with early outbreak phylodynamics (preprint)

Understanding how SARS-CoV-2 entered the human population, thereby causing the COVID-19 pandemic, is one of the most urgent questions in science today. Two hypotheses are widely acknowledged as being most likely to explain the pandemic's origin in late 2019: (i) the natural origin hypothesis that one or more cross-species transmissions from animals into humans occurred, most likely at the Huanan Seafood Market in Wuhan, China; (ii) the laboratory origin hypothesis, that scientific research activities led to the unintentional leak of SARS-CoV-2 from a laboratory into the general population. A recent analysis of SARS-CoV-2 genomes by Pekar et al. [Science 377:960-966 (2022)] claims to establish at least two separate spillover events from animals into humans, thus claiming to provide strong evidence for the natural origin hypothesis. However, here we use outbreak simulations to show that the findings of Pekar et al. are heavily impacted by two methodological artifacts: the dubious exclusion of informative SARS-CoV-2 genomes, and their reliance on unrealistic phylodynamic models of SARS-CoV-2. Absent models that incorporate these effects, one cannot conclude multiple SARS-CoV-2 spillovers into humans. Our results cast doubt on a primary point of evidence in favor of the natural origin hypothesis.

Further analysis of metagenomic datasets containing GD and GX pangolin CoVs indicates widespread contamination, undermining pangolin host attribution (preprint)

The only animals other than bats reported to have been infected with SARS-CoV-2-related coronaviruses (SARS2r-CoVs) prior to the COVID-19 pandemic are pangolins. In early 2020 multiple papers reported the identification of two clades of SARS2r-CoVs, GD and GX, infecting pangolins. However the RNA-Seq datasets supporting pangolin genome assembly were widely contaminated, contained synthetic vectors or were heavily enriched or filtered with little but coronavirus sequences left in the datasets. Here we investigate two pangolin fecal samples sequenced by Li et al. (2021) provided in support of GD PCoV infection of pangolins in Guangdong and find the read distribution consistent with PCR amplicon contamination and SARS-CoV-2 contamination, and further identify the presence of synthetic plasmid sequences. We also build upon our previous work to further analyze the dataset GX/P3B by Lam et al. (2020), which is the only non enriched/heavily filtered pangolin tissue dataset sequenced by Lam et al. (2020). We identify synthetic vectors and confirm human genomic origin samples in the dataset. Finally, we find human mitochondrial sequences in all pangolin organ datasets and mouse and tiger mitochondrial sequences in selected pangolin organ datasets sequenced by Liu et al. (2019). We infer that human and mouse genomic origin sequences were probably sourced from contamination prior to sequencing, while tiger origin sequence contamination may have occurred due to index hopping during sequencing. These observations are problematic for attributing pangolins as SARS2r-CoV hosts in the datasets examined. The forensic methods developed and used here can be applied to examine any third party SRA data sets.

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 (preprint)

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.

Nipah virus vector sequences in COVID-19 patient samples sequenced by the Wuhan Institute of Virology (preprint)

We report the detection of Nipah virus in an infectious clone format, a BSL4-level pathogen and CDC-designated Bioterrorism Agent, in raw RNA-Seq sequencing reads deposited by the Wuhan Institute of Virology (WIV) produced from five December 2019 patients infected with SARS-CoV-2. Research involving Nipah infectious clones has never been reported to have occured at the WIV. These patient samples have been previously reported to contain reads from several other viruses: Influenza A, Spodoptera frugiperda rhabdovirus and Nipah. Previous authors have interpreted the presence of these virus sequences as indicative of co-infections of the patients in question by these pathogens or laboratory contamination. However, our analysis shows that NiV genes are encapsulated in synthetic vectors, which we infer was for assembly of a NiV infectious clone. In particular, we document the finding of internal N, P-V-W-C and L protein coding sequences as well as coverage of the G and F genes. Furthermore, the format of Hepatitis D virus ribozyme and T7 terminator downstream of the 5-prime end of the NiV sequence is consistent with truncation required at the end of the genome for a full length infectious clone. This indicates that research at WIV was being conducted on an assembled NiV infectious clone. Contamination of patient sequencing reads by an infectious NiV clone of the highly pathogenic Bangladesh strain could indicate a significant breach of BSL-4 protocols. We call on WIV to explain the purpose of this research on infectious clones of Nipah Virus, the full chronology of this work, and to explain how and at what stage of sample preparation this contamination occurred.

Analysis of pangolin metagenomic datasets reveals significant contamination, raising concerns for pangolin CoV host attribution (preprint)

Metagenomic datasets from pangolin tissue specimens have previously yielded SARS-related coronaviruses which show high homology in their receptor binding domain to SARS-CoV-2, suggesting a potential zoonotic source for this feature of the human virus, possibly via recombination (Liu et al. 2019, Lam et al. 2020, Xiao et al. 2020, Liu et al. 2020). Here we re-examine these published datasets. We report that only a few pangolin samples were found to contain coronavirus reads, and even then in low abundance, while other non-pangolin hosted viruses were present in higher abundance. We also discovered extensive contamination with human, rodent, and other mammalian gene sequences, which was a surprising finding. Furthermore, we uncovered a number of pangolin CoV sequences embedded in standard laboratory cloning vectors, which suggests the pangolin specimens could have been contaminated with sequences derived from synthetic biology experiments. Finally, we discover a third pangolin dataset (He et al. 2022) with low levels of SARSr-CoV sequences and unambiguous extensive contamination of several pangolin samples. For these reasons, we find it unlikely that the pangolins in question had a coronavirus infection while alive, and all current versions of the cited papers claiming a zoonotic infection of pangolins with a SARS-r CoV require substantial corrections and should be retracted until such corrections are made.

An appeal for an open scientific debate about the proximal origin of SARS-CoV-2 (preprint)

One year after the onset of the COVID-19 pandemic, the origin of SARS-CoV-2 still eludes humanity. Early publications firmly stated that the virus was of natural origin, and the possibility that the virus might have escaped from a lab was discarded in most subsequent publications. However, based on a re-analysis of the initial arguments, highlighted by the current knowledge about the virus, we show that the natural origin is not supported by conclusive arguments, and that a lab origin cannot be formally discarded. We call for an opening of peer-reviewed journals to a rational, evidence-based and prejudice-free evaluation of all the reasonable hypotheses about the virus' origin. We advocate that this debate should take place in the columns of renowned scientific journals, rather than being left to social media and newspapers.

Unexpected novel Merbecovirus discoveries in agricultural sequencing datasets from Wuhan, China (preprint)

In this study we document the unexpected discovery of multiple coronaviruses and a BSL-3 pathogen in agricultural cotton and rice sequencing datasets. In particular, we have identified a novel HKU5-related Merbecovirus in a cotton dataset sequenced by the Huazhong Agricultural University in 2017. We have also found an infectious clone sequence containing a novel HKU4-related Merbecovirus related to MERS coronavirus in a rice dataset sequenced by the Huazhong Agricultural University in early 2020. Another HKU5-related Merbecovirus, as well as Japanese encephalitis virus, were identified in a cotton dataset sequenced by the Huazhong Agricultural University in 2018. An HKU3-related Betacoronavirus was found in a Mus musculus sequencing dataset from the Wuhan Institute of Virology in 2017. Finally, a SARS-WIV1-like Betacoronavirus was found in a rice dataset sequenced by the Fujian Agriculture and Forestry University in 2017. Using the contaminating reads we have extracted from the above datasets, we were able to assemble complete genomes of two novel coronaviruses which we disclose herein. In light of our findings, we raise concerns about biosafety protocol breaches, as indicated by our discovery of multiple dangerous human pathogens in agricultural sequencing laboratories in Wuhan and Fouzou City, China.

The bat coronavirus RmYN02 is characterized by a 6-nucleotide deletion at the S1/S2 junction, and its claimed PAA insertion is highly doubtful (preprint)

Zhou et al. reported the discovery of RmYN02, a strain closely related to SARS-CoV-2, which is claimed to contain a natural PAA amino acid insertion at the S1/S2 junction of the spike protein at the same position of the PRRA insertion that has created a polybasic furin cleavage site in SARS-CoV-2. The authors support with their findings the theory that the furin cleavage site insertion present in SARS-CoV-2 is natural. Because no nucleotide alignment with closely related strains of the region coding for the supposed insertion is provided by Zhou et al., we have applied several alignment algorithms to search for the most parsimonious alignments. We conclude that RmYN02 does not contain an insertion at the S1/S2 junction when compared to its closest relatives at the nucleotide level, but rather a 6-nucleotide deletion and that the claimed PAA insertion is more likely to be the result of mutations. A close examination of RmYN02 sequencing records and assembly methods is wishful. In conclusion, SARS-CoV-2, with its 12-nucleotide insertion at the S1/S2 junction remains unique among its sarbecovirus relatives.