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Coronavirus was bioengineered and leaked discussion by Dr Paul Cottrell


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Coronavirus (proof of a bio-weapon?) by Dr. Paul Cottrell


A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence

Published: 09 November 2015
Zhengli-Li Shi & more
Abstract

The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome (MERS)-CoV underscores the threat of cross-species transmission events leading to outbreaks in humans. Here we examine the disease potential of a SARS-like virus, SHC014-CoV, which is currently circulating in Chinese horseshoe bat populations1. Using the SARS-CoV reverse genetics system2, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse-adapted SARS-CoV backbone. The results indicate that group 2b viruses encoding the SHC014 spike in a wild-type backbone can efficiently use multiple orthologs of the SARS receptor human angiotensin converting enzyme II (ACE2), replicate efficiently in primary human airway cells and achieve in vitro titers equivalent to epidemic strains of SARS-CoV.

Angiotensin-converting enzyme 2 (ACE2) proteins of different bat species confer variable susceptibility to SARS-CoV
entry

Published: 22 June 2010 Yuxuan Hou, Cheng Peng, Meng Yu, Yan Li, Zhenggang Han, Fang Li, Lin-Fa Wang & Zhengli Shi
Abstract

The discovery of SARS-like coronavirus in bats suggests that bats could be the natural reservoir of SARS-CoV. However, previous studies indicated the angiotensin-converting enzyme 2 (ACE2) protein, a known SARS-CoV receptor, from a horseshoe bat was unable to act as a functional receptor for SARS-CoV. Here, we extended our previous study to ACE2 molecules from seven additional bat species and tested their interactions with human SARS-CoV spike protein using both HIV-based pseudotype and live SARS-CoV infection assays. The results show that ACE2s of Myotis daubentoni and Rhinolophus sinicus support viral entry mediated by the SARS-CoV S protein, albeit with different efficiency in comparison to that of the human ACE2.
Difference in Receptor Usage between Severe Acute Respiratory Syndrome (SARS) Coronavirus and SARS-Like Coronavirus of Bat Origin
Wuze Ren, Xiuxia Qu, Wendong Li, Zhenggang Han, Meng Yu, Peng Zhou, Shu-Yi Zhang, Lin-Fa Wang, Hongkui Deng, Zhengli Shi
ABSTRACT

Severe acute respiratory syndrome (SARS) is caused by the SARS-associated coronavirus (SARS-CoV), which uses angiotensin-converting enzyme 2 (ACE2) as its receptor for cell entry. A group of SARS-like CoVs (SL-CoVs) has been identified in horseshoe bats. SL-CoVs and SARS-CoVs share identical genome organizations and high sequence identities, with the main exception of the N terminus of the spike protein (S), known to be responsible for receptor binding in CoVs. In this study, we investigated the receptor usage of the SL-CoV S by combining a human immunodeficiency virus-based pseudovirus system with cell lines expressing the ACE2 molecules of human, civet, or horseshoe bat. In addition to full-length S of SL-CoV and SARS-CoV, a series of S chimeras was constructed by inserting different sequences of the SARS-CoV S into the SL-CoV S backbone. Several important observations were made from this study. First, the SL-CoV S was unable to use any of the three ACE2 molecules as its receptor. Second, the SARS-CoV S failed to enter cells expressing the bat ACE2. Third, the chimeric S covering the previously defined receptor-binding domain gained its ability to enter cells via human ACE2, albeit with different efficiencies for different constructs. Fourth, a minimal insert region (amino acids 310 to 518) was found to be sufficient to convert the SL-CoV S from non-ACE2 binding to human ACE2 binding, indicating that the SL-CoV S is largely compatible with SARS-CoV S protein both in structure and in function. The significance of these findings in relation to virus origin, virus recombination, and host switching is discussed.

The S proteins of CoVs play a key role in virus entry into host cells, including binding to host cell receptors and membrane fusion (4, 10, 24). Angiotensin-converting enzyme 2 (ACE2) has been identified as the functional receptor of SARS-CoV, and the molecular interaction between ACE2 and the SARS-CoV S protein has been well characterized (27, 28, 31, 42). A 193-residue fragment (amino acids [aa] 318 to 510) in the SARS-CoV S protein was demonstrated to be the minimal receptor-binding domain (RBD) which alone was able to efficiently bind to ACE2 (1, 42a, 45). Furthermore, it was shown that minor changes in amino acid residues of the receptor-binding motif (RBM) of SARS-CoV S protein could abolish the entry of SARS-CoV into cells expressing human ACE2 (huACE2) (7, 31). In the corresponding RBD region of the SL-CoV S proteins, there is significant sequence divergence from those of the SARS-CoV S proteins, including two deletions of 5 and 12 or 13 aa. From crystal-structural analysis of the S-ACE2 complex, it was predicted that the S protein of SL-CoV is unlikely to use huACE2 as an entry receptor (30), although this has never been experimentally proven due to the lack of live SL-CoV isolates. Whether it is possible to construct an ACE2-binding SL-CoV S protein by replacing the RBD with that from SARS-CoV S proteins is also unknown.

In this study, a human immunodeficiency virus (HIV)-based pseudovirus system was employed to address these issues.
MATERIALS AND METHODS

A MAb against p24 of HIV was generated by the HIV group of the Wuhan Institute of Virology

Construction of expression plasmids.The construction of a codon-optimized spike (S) protein gene of SARS-CoV BJ01 (BJ01-S) in pcDNA3.1(+) was described previously (34, 46). The full-length S gene of a bat SL-CoV (Rp3) was cloned by PCR amplification from cDNA prepared using fecal samples from an R. pearsonii bat positive for SL-CoV (29).

Cloning of ACE2 genes and establishment of ACE2-expressing cell lines.The coding region for the ACE2 homolog in the horseshoe bat R. pearsonii was obtained from the intestine of a bat infected with SL-CoV by PCR using the following primers:

Construction and purification of pseudoviruses.HIV-based pseudoviruses were prepared as described previously (37).

GenBank accession number.The sequence of the R. pearsonii ACE2 coding region has been deposited in GenBank under the accession number EF569964.

Cloning of bat ACE2 and its functional expression in HeLa cells.The ACE2 gene homolog amplified from the horseshoe bat R. pearsonii(RpACE2) codes for an 805-aa protein that is identical in size to the ACE2 proteins of humans and civets. Sequence alignment indicated that the bat RpACE2 molecule is similar to the ACE2 proteins of other mammals and has an amino acid sequence identity of 81% to huACE2 and pcACE2 and 77% to mouse and rat ACE2 proteins. As shown in Fig. 1, the major sequence variation is located in the N-terminal region. For the 18 residues of huACE2 known to make direct contact with the SARS-CoV S protein (27), there are seven changes in RpACE2 compared to the cognate human protein: Q24R (Q in huACE2 and R in RpACE2 at aa 24), Y41H, Q42E, M82D, Q325E, E329N, and G354D (Fig. 1).

*Corresponding author. Mailing address for Z. Shi: State key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China. Phone: (86-27)-87197240. Fax: (86-27)-87197240. E-mail: [email protected]. Mailing address for L.-F. Wang: CSIRO Livestock Industries, Australian Animal Health Laboratory, P.O. Bag 24, Geelong, Victoria 3220, Australia. Phone: (61-3)-52275121. Fax: (61-3)-52275555. E-mail: [email protected]

† W.R. and X.Q. contributed equally to this work.

‡ Present address: School of Life Science, Heilongjiang University, Harbin, 150080, China.

▿ Published ahead of print on 12 December 2007.


On the Origins of the 2019-nCoV Virus, Wuhan, China
https://jameslyonsweiler.com/2020/01/30/on-the-origins-of-the-2019-ncov-virus-wuhan-china/

RECOMBINATION technology has been in use in molecular virology since the 1980’s. The structure of the 2019-NCoV virus genome provides a very strong clue on the likely origin of the virus.

Unlike other related coronaviruses, the 2019-nCoV virus has a unique sequence about 1,378 bp (nucleotide base pairs) long that is not found in related coronaviruses.
Irrefutable: The coronavirus was engineered by scientists in a lab using well documented genetic engineering vectors that leave behind a “fingerprint”

https://www.naturalnews.com/2020-02-03-the-coronavirus-was-engineered-by-scientists-in-a-lab.html
https://patents.google.com/patent/CN1276777C/en?q=pshuttle-SN&oq=pshuttle-SN
pshuttle-SN;

SARS vaccine of adenovirus vector and preparation method, application of coronavirus S gene

Abstract
translated from Chinese
The present invention belongs to the field of genetic engineering, particularly relates to adenoviral vector SARS vaccines, their preparation and coronavirus S genes in SARS (SARS) on vaccines for the prophylaxis. By means of biological engineering, the coronavirus S gene in combination with deficient recombinant adenovirus, the protective immunogen protein or polypeptide expressed therein, through expansion culture, purification, and formulation to prepare a mucosal immunogenicity can cause the gene vaccine, respiratory mucosal immune response induced by the body to produce antibodies against the virus infection. Specific conditions of the present invention, compared with conventional inactivated virus particle vaccine, safe, easy to use, without limitation intramuscular, have broad clinical applications.