In 2015, Shi Zhengli was involved in an investigation of bat coronaviruses, specifically gain of function experiments involving both the SARS and bat coronaviruses, a joint research of University of North Carolina and Wuhan Institute of Virology, with Ralph S. Baric as principal investigator.
The 2015 Gain-of-Function experiment by Shi and others was famously known to have created a so-called Chimeric or Hybrid Virus and a version of which was believed to have been leaked (either accidentally or deliberately) from Shi's main lab the Wuhan Institute of Virology in late 2019. That lethal and extremely contagious virus is now called COVID-19 a.k.a. Wuhan Corona Virus.
That same year the generous funding for the project in the US had been paused due to the moratorium on risky (Gain-of-Function) virology studies with influenza, MERS & SARS viruses, announced by the US government that year. But it is obvious now that despite US Moratorium Chinese Communist Party let the Chinese scientists to continue extremely dangerous G-o-F experiments.
(Following is the extracts from the research report, of that 2015 GOF Experiment, written by Shi Zhengli herself and her American co-authors and published in the NATURE magazine 2015-October.)
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 populations. Using the SARS-CoV reverse genetics system, 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.
On the basis of these findings, we synthetically re-derived an infectious full-length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo. Our work suggests a potential risk of SARS-CoV re-emergence from viruses currently circulating in bat populations.
Vineet D Menachery, Boyd L Yount Jr, Kari Debbink, Sudhakar Agnihothram, Lisa E Gralinski, Jessica A Plante, Rachel L Graham, Trevor Scobey, Ralph S Baric, Eric F Donaldson, Scott H Randell, Antonio Lanzavecchia, Wayne A Marasco, Xing-Yi Ge & Zheng-Li Shi.
The emergence of SARS-CoV heralded a new era in the cross-species transmission of severe respiratory illness with globalization leading to rapid spread around the world and massive economic impact. Since then, several strains—including influenza A strains H5N1, H1N1 and H7N9 and MERS-CoV—have emerged from animal populations, causing considerable disease, mortality and economic hardship for the afflicted regions.
Although public health measures were able to stop the SARS-CoV outbreak4, recent metagenomics studies have identified sequences of closely related SARS-like viruses circulating in Chinese bat populations that may pose a future threat. However, sequence data alone provides minimal insights to identify and prepare for future prepandemic viruses.
Therefore, to examine the emergence potential (that is, the potential to infect humans) of circulating bat CoVs, we built a chimeric virus encoding a novel, zoonotic CoV spike protein—from the RsSHC014-CoV sequence that was isolated from Chinese horseshoe bats1—in the context of the SARS-CoV mouse-adapted backbone.
The sequences of SHC014 and the related RsWIV1-CoV show that these CoVs are the closest relatives to the epidemic SARS-CoV strains; however, there are important differences in the 14 residues that bind human ACE2, the receptor for SARS-CoV, including the five that are critical for host range: Y442, L472, N479, T487 and Y491.
In WIV1, three of these residues vary from the epidemic SARS-CoV Urbani strain, but they were not expected to alter binding to ACE2. This fact is confirmed by both pseudotyping experiments that measured the ability of lentiviruses encoding WIV1 spike proteins to enter cells expressing human ACE2 and by in vitro replication assays of WIV1-CoV.
In contrast, 7 of 14 ACE2-interaction residues in SHC014 are different from those in SARS-CoV, including all five residues critical for host range. These changes, coupled with the failure of pseudotyped lentiviruses expressing the SHC014 spike to enter cells, suggested that the SHC014 spike is unable to bind human ACE2.
However, similar changes in related SARS-CoV strains had been reported to allow ACE2 binding, suggesting that additional functional testing was required for verification. Therefore, we synthesized the SHC014 spike in the context of the replication-competent, mouse-adapted SARS-CoV backbone (we hereafter refer to the chimeric CoV as SHC014-MA15) to maximize the opportunity for pathogenesis and vaccine studies in mice.
Despite predictions from both structure-based modeling and pseudotyping experiments, SHC014-MA15 was viable and replicated to high titers in Vero cells. Similarly to SARS, SHC014-MA15 also required a functional ACE2 molecule for entry and could use human, civet and bat ACE2 orthologs.
To test the ability of the SHC014 spike to mediate infection of the human airway, we examined the sensitivity of the human epithelial airway cell line Calu-3 2B4 to infection and found robust SHC014-MA15 replication, comparable to that of SARS-CoV Urbani. To extend these findings, primary human airway epithelial (HAE) cultures were infected and showed robust replication of both viruses.
Together, the data confirm the ability of viruses with the SHC014 spike to infect human airway cells and underscore the potential threat of cross-species transmission of SHC014-CoV.
Viruses, cells, in vitro infection and plaque assays. Wild-type SARS-CoV (Urbani), mouse-adapted SARS-CoV (MA15) and chimeric SARS-like CoVs were cultured on Vero E6 cells (obtained from United States Army Medical Research Institute of Infectious Diseases), grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco, CA) and 5% fetal clone serum (FCS) (Hyclone, South Logan, UT) along with antibiotic/antimycotic (Gibco, Carlsbad, CA).
DBT cells (Baric laboratory, source unknown) expressing ACE2 orthologs have been previously described for both human and civet; bat Ace2 sequence was based on that from Rhinolophus leschenaulti, and DBT cells expressing bat Ace2 were established as described previously8.
Inserting S Spike-proteins into SARS Bat Virus to make new
Virus as original SARS spikes are unable to latch onto the
ACE2 receptors of human cells and thus not contagious.
Growth curves in Vero E6, DBT, Calu-3 2B4 and primary human airway epithelial cells were performed as previously described. None of the working cell line stocks were authenticated or tested for mycoplasma recently, although the original seed stocks used to create the working stocks are free from contamination.
Human lungs for HAE cultures were procured under University of North Carolina at Chapel Hill Institutional Review Board–approved protocols. HAE cultures represent highly differentiated human airway epithelium containing ciliated and non-ciliated epithelial cells as well as goblet cells. The cultures are also grown on an air-liquid interface for several weeks before use, as previously described.
Briefly, cells were washed with PBS and inoculated with virus or mock-diluted in PBS for 40 min at 37 °C. After inoculation, cells were washed three times and fresh medium was added to signify time '0'. Three or more biological replicates were harvested at each described time point. No blinding was used in any sample collections nor were samples randomized.
All virus cultivation was performed in a biosafety level (BSL) 3 laboratory with redundant fans in the biosafety cabinets, as described previously by our group. All personnel wore powered air purifying respirators (Breathe Easy, 3M) with Tyvek suits, aprons and booties and were double-gloved.
Both wild-type and chimeric viruses were derived from either SARS-CoV Urbani or the corresponding mouse-adapted (SARS-CoV MA15) infectious clone (ic) as previously described. Plasmids containing spike sequences for SHC014 were extracted by restriction digest and ligated into the E and F plasmid of the MA15 infectious clone.
The clone was designed and purchased from Bio Basic as six contiguous cDNAs using published sequences flanked by unique class II restriction endonuclease sites (BglI). Thereafter, plasmids containing wild-type, chimeric SARS-CoV and SHC014-CoV genome fragments were amplified, excised, ligated and purified.
In vitro transcription reactions were then preformed to synthesize full-length genomic RNA, which was transfected into Vero E6 cells as previously described. The medium from transfected cells was harvested and served as seed stocks for subsequent experiments.
Chimeric and full-length viruses were confirmed by sequence analysis before use in these studies. Synthetic construction of chimeric mutant and full-length SHC014-CoV was approved by the University of North Carolina Institutional Biosafety Committee and the Dual Use Research of Concern committee.
Female, 10-week-old and 12-month-old BALB/cAnNHsD mice were ordered from Harlan Laboratories. Mouse infections were done as previously described. Briefly, animals were brought into a BSL3 laboratory and allowed to acclimate for 1 week before infection.
For infection and live-attenuated virus vaccination, mice were anesthetized with a mixture of ketamine and xylazine and infected intra-nasally, when challenged, with 50 μl of phosphate-buffered saline (PBS) or diluted virus with three or four mice per time point, per infection group per dose as described in the figure legends.
For individual mice, notations for infection including failure to inhale the entire dose, bubbling of inoculum from the nose, or infection through the mouth may have led to exclusion of mouse data at the discretion of the researcher; post-infection, no other pre-established exclusion or inclusion criteria are defined. No blinding was used in any animal experiments, and animals were not randomized.
For vaccination, young and aged mice were vaccinated by footpad injection with a 20-μl volume of either 0.2 μg of double-inactivated SARS-CoV vaccine with alum or mock PBS; mice were then boosted with the same regimen 22 d later and challenged 21 d thereafter. For all groups, as per protocol, animals were monitored daily for clinical signs of disease (hunching, ruffled fur and reduced activity) for the duration of the experiment.
Mice that lost greater than 30% of their starting body weight were immediately sacrificed as per protocol. Any mouse deemed to be moribund or unlikely to recover was also humanely sacrificed at the discretion of the researcher. Euthanasia was performed using an isoflurane overdose and death was confirmed by cervical dislocation.
Biosafety and biosecurity:
Reported studies were initiated after the University of North Carolina Institutional Biosafety Committee approved the experimental protocol (Project Title: Generating infectious clones of bat SARS-like CoVs; Lab Safety Plan ID: 20145741; Schedule G ID: 12279).
These studies were initiated before the US Government Deliberative Process Research Funding Pause on Selected Gain-of-Function Research Involving Influenza, MERS and SARS Viruses (http://www.phe.gov/s3/dualuse/Documents/gain-of-function.pdf). This paper has been reviewed by the funding agency, the NIH. Continuation of these studies was requested, and this has been approved by the NIH.
30 March 2020 Nature Editors’ note: We are aware that this article is being used as the basis for unverified theories that the novel coronavirus causing COVID-19 was engineered. There is no evidence that this is true; (there also is no evidence that this is not true;) scientists believe that an animal is the most likely source of the coronavirus.
20 November 2015: In the version of this article initially published online, the authors omitted to acknowledge a funding source, USAID-EPT-PREDICT funding from EcoHealth Alliance, to Z.-L.S. The error has been corrected for the print, PDF and HTML versions of this article.
(Blogger's Notes: Just a few years back so many micro-biologists were so alarmingly disturbed by some rebellious micro-biologists, like Yoshiro Kawaoka and Ron Fouchier, creating influenza virus with man-made pandemic potentials that they even forced Obama's US government to order a strict moratorium on so-called Gain-of-Function experiments.
May our gods have mercy on us god-fearing humans, for the godless Chinese Communists with Shi Zhengli's 'Wuhan Corona Virus' won't have one, and they have ended the humanity as we know it.)
Related posts at following links:
Pandemic Potentials of Gain-of-Function Experiments.
Gain-of-Function Experiments Created the Wuhan Corona Virus.
Game-of-Functions Experiment: Fears of Man-made Pandemic (2014).
Evil Scientists Creating new viruses with Pandemic Potential.
Wuhan Corona Virus: Genetically Engineered To Infect Humans.