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1. PROPOSED FORENSIC INVESTIGATION OF WUHAN LABORATORIES

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This is the first part of a series of reports based on our previously unpublished investigations into the origins of SARS-COV-2. We wish to thank all the independent researchers who have contributed to this investigation, especially members of the DRASTIC Collective, many of whom wish to remain anonymous for reasons of security and privacy. Two appendices cannot be uploaded to ResearchGate but are available for download via Zenodo: Appendix 14 FAO_LMT_CoreR.xlsx https://zenodo.org/record/4640384/files/Appendix%2014%20FAO_LMT_CoreR.xlsx?download=1 Appendix 15 FAO LMT_SAFETY.xlsx https://zenodo.org/record/4640384/files/Appendix%2015%20FAO%20LMT_SAFETY.xlsx?download=1
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Abstract: RaTG13 is the closest related coronavirus genome phylogenetically to SARS-CoV-2, consequently understanding its provenance is of key importance to understanding the origin of the COVID-19 pandemic. The RaTG13 NGS dataset is attributed to a fecal swab from the intermediate horseshoe bat Rhinolophus affinis. However, sequence analysis reveals that this is unlikely. Metagenomic analysis using Metaxa2 shows that only 10.3 % of small subunit (SSU) rRNA sequences in the dataset are bacterial, inconsistent with a fecal sample, which are typically dominated by bacterial sequences. In addition, the bacterial taxa present in the sample are inconsistent with fecal material. Assembly of mitochondrial SSU rRNA sequences in the dataset produces a contig 98.7 % identical to R.affinis mitochondrial SSU rRNA, indicating that the sample was generated from this or a closely related species. 87.5 % of the NGS reads map to the Rhinolophus ferrumequinum genome, the closest bat genome to R.affinis available. In the annotated genome assembly, 62.2 % of mapped reads map to protein coding genes. These results clearly demonstrate that the dataset represents a Rhinolophus sp. transcriptome, and not a fecal swab sample. Overall, the data show that the RaTG13 dataset was generated by the Wuhan Institute of Virology (WIV) from a transcriptome derived from Rhinolophus sp. tissue or cell line, indicating that RaTG13 was in live culture. This raises the question of whether the WIV was culturing additional unreported coronaviruses closely related to SARS-CoV-2 prior to the pandemic. The implications for the origin of the COVID-19 pandemic are discussed.
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This is the fourth part of a series of reports based on previously unpublished investigations into the origins of SARS-COV-2 by William Bostickson and Yvette Ghannam, which took place in 2020. This report also contains novel insights from ongoing joint investigations with Gilles Demaneuf from the DRASTIC collective in 2021, but it is worth noting that the majority of the content was analysed in 2020.
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A coronavirus (CoV) commonly known as SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and causing COVID-19 (coronavirus disease of 2019) has become a pandemic following an outbreak in Wuhan. Although mutations in the SARS-CoV-2 spike glycoprotein (SGP) are obvious from comparative genome studies, the novel infectious nature of the virus, its new varients detected in the UK, and outside and recovery–death ratios of COVID-19 inspired us to review the mechanisms of the infection, replication, release, and transmission of progeny virions and the immune response in the host cell. In addition to the specificity of SARS-CoV-2 binding to angiotensin-converting enzyme 2 receptor and transmembrane protease serine 2, the varied symptoms and severity of the infection by the original and mutated forms of the virus suggest the significance of correlating the host innate and adaptive immunity with the binding of the virus to the mannose receptor via lipopolysaccharides (LPSs), toll-like receptors via LPS/proteins/RNA, and sialic acid (Sia) via hemagglutinin, or sugar-acid segments of glycans. HA-to-Sia binding is considered based on the innate Sia N-acetylneuraminic acid and the acquired Sia N-glycolylneuraminic acid in the epithelial cells and the sialidase/neuraminidase- or esterase-hydrolyzed release and transmission of CoVs. Furthermore, the cytokine storms common to aged humans infected with SARS-CoV-2 and aged macaques infected with SARS-CoV encourage us to articulate the mechanism by which the nuclear capsid protein and RNAs bypass the pattern recognition-induced secretion of interferons (IFNs), which stimulate IFN genes through the Janus-activated kinase-signal transducer and activator of a transcription pathway, leading to the secretion of antiviral proteins such as myxovirus resistance protein A/B. By considering the complexities of the structure, and the infectious nature of the virus and the structures and functions of the molecules involved in CoV infection, replication, and immune response, a new interface among virology, immunology, chemistry, imaging technology, drug delivery, and nanoscience is proposed and will be developed. This interface can be an essential platform for researchers, technologists, and physicians to collaborate and develop vaccines and medicines against COVID-19 and other pandemics in the future. Scientists in Japan have reviewed how nanoscience is helping us understand infection with SARS-CoV-2, the virus responsible for Covid-19, and the immune response it produces. The coronavirus pandemic has driven international scientific collaboration to identify treatments and develop a vaccine, not only between virologists and immunologists but also with researchers from a broad range of other disciplines including chemists, physicists and materials scientists. Vasudevanpillai Biju from Hokkaido University, Sapporo, and colleagues have reviewed the ongoing research at the interface of infectious diseases, biological chemistry and nanoscience aimed at answering key questions on how the virus functions. The authors summarize the use of nanomaterials in imaging techniques, vaccine development and drug delivery, while investigating problems associated with the toxicity of nanomaterials. Understanding these molecular interactions will help to fight this and future pandemics. Despite the hopeful signs of progress of COVID-19 vaccine development and vaccination, the highly infectious nature and mutations of SARS-CoV-2 are warnings of an infighting annual revival of the virus. This article clarifies the complexities of COVID-19 by referring to the molecular-level mechanisms of the infection, immune response, replication, and transmission of SARS-CoV-2, which are essential during the development of an effective vaccine or a drug to fight the pandemic. Furthermore, this article underscores the significance of an interface among chemistry, nanoscience, cell biology, immunology, and virology to resolve the challenges of COVID-19.
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SARS Coronavirus 2 (SARS-CoV-2) emerged in late 2019, leading to the Coronavirus Disease 2019 (COVID-19) pandemic that continues to cause significant global mortality in human populations. Given its sequence similarity to SARS-CoV, as well as related coronaviruses circulating in bats, SARS-CoV-2 is thought to have originated in Chiroptera species in China. However, whether the virus spread directly to humans or through an intermediate host is currently unclear, as is the potential for this virus to infect companion animals, livestock, and wildlife that could act as viral reservoirs. Using a combination of surrogate entry assays and live virus, we demonstrate that, in addition to human angiotensin-converting enzyme 2 (ACE2), the Spike glycoprotein of SARS-CoV-2 has a broad host tropism for mammalian ACE2 receptors, despite divergence in the amino acids at the Spike receptor binding site on these proteins. Of the 22 different hosts we investigated, ACE2 proteins from dog, cat, and cattle were the most permissive to SARS-CoV-2, while bat and bird ACE2 proteins were the least efficiently used receptors. The absence of a significant tropism for any of the 3 genetically distinct bat ACE2 proteins we examined indicates that SARS-CoV-2 receptor usage likely shifted during zoonotic transmission from bats into people, possibly in an intermediate reservoir. Comparison of SARS-CoV-2 receptor usage to the related coronaviruses SARS-CoV and RaTG13 identified distinct tropisms, with the 2 human viruses being more closely aligned. Finally, using bioinformatics, structural data, and targeted mutagenesis, we identified amino acid residues within the Spike–ACE2 interface, which may have played a pivotal role in the emergence of SARS-CoV-2 in humans. The apparently broad tropism of SARS-CoV-2 at the point of viral entry confirms the potential risk of infection to a wide range of companion animals, livestock, and wildlife.
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