Zhu “SARS-COV-2” Paper (2020)

“[We show] an image of sedimented virus particles, not purified ones.”

-Replying Author: Wenjie Tan

https://off-guardian.org/2020/06/27/covid19-pcr-tests-are-scientifically-meaningless/

The Zhu study gets the infamous distinction for not only acknowledging that they did not fulfill Koch’s Postulates, the logic-based criteria needed to be met in order to prove a microorganism causes disease, but also for admitting that they did not purify (free from foreign material, contaminants, pollutants) their “isolates.” This means that their “isolate” contains millions of similar and/or identical particles which could also be their culprit…or none of the particles in their sample could be. That being the case, these researchers can hardly claim that they isolated (separated from everything else) a “virus” at all. They present similar evidence and findings as the earlier Zhou study yet neither of them were able to satisfy the proper scientific criteria needed to prove a new “virus” exists nor that it can also cause disease. Highlights below:

A Novel Coronavirus from Patients with Pneumonia in China, 2019

VIRAL DIAGNOSTIC METHODS

Four lower respiratory tract samples, including bronchoalveolar-lavage fluid, were collected from patients with pneumonia of unknown cause who were identified in Wuhan on December 21, 2019, or later and who had been present at the Huanan Seafood Market close to the time of their clinical presentation. Seven bronchoalveolar-lavage fluid specimens were collected from patients in Beijing hospitals with pneumonia of known cause to serve as control samples.”

ISOLATION OF VIRUS

“Bronchoalveolar-lavage fluid samples were collected in sterile cups to which virus transport medium was added. Samples were then centrifuged to remove cellular debris. The supernatant was inoculated on human airway epithelial cells,13 which had been obtained from airway specimens resected from patients undergoing surgery for lung cancer and were confirmed to be special-pathogen-free by NGS.14

“Prior to infection, apical surfaces of the human airway epithelial cells were washed three times with phosphate-buffered saline; 150 μl of supernatant from bronchoalveolar-lavage fluid samples was inoculated onto the apical surface of the cell cultures. After a 2-hour incubation at 37°C, unbound virus was removed by washing with 500 μl of phosphate-buffered saline for 10 minutes; human airway epithelial cells were maintained in an air–liquid interface incubated at 37°C with 5% carbon dioxide. Every 48 hours, 150 μl of phosphate-buffered saline was applied to the apical surfaces of the human airway epithelial cells, and after 10 minutes of incubation at 37°C the samples were harvested. Pseudostratified mucociliary epithelium cells were maintained in this environment; apical samples were passaged in a 1:3 diluted vial stock to new cells. The cells were monitored daily with light microscopy, for cytopathic effects, and with RT-PCR, for the presence of viral nucleic acid in the supernatant. After three passages, apical samples and human airway epithelial cells were prepared for transmission electron microscopy.

TRANSMISSION ELECTRON MICROSCOPY

Supernatant from human airway epithelial cell cultures that showed cytopathic effects was collected, inactivated with 2% paraformaldehyde for at least 2 hours, and ultracentrifuged to sediment virus particles. The enriched supernatant was negatively stained on film-coated grids for examination. Human airway epithelial cells showing cytopathic effects were collected and fixed with 2% paraformaldehyde–2.5% glutaraldehyde and were then fixed with 1% osmium tetroxide dehydrated with grade ethanol embedded with PON812 resin. Sections (80 nm) were cut from resin block and stained with uranyl acetate and lead citrate, separately. The negative stained grids and ultrathin sections were observed under transmission electron microscopy.

VIRAL GENOME SEQUENCING

“RNA extracted from bronchoalveolar-lavage fluid and culture supernatants was used as a template to clone and sequence the genome.”

Multiple-sequence alignment of the 2019-nCoV and reference sequences was performed with the use of Muscle.”

DETECTION AND ISOLATION OF A NOVEL CORONAVIRUS

“Three bronchoalveolar-lavage samples were collected from Wuhan Jinyintan Hospital on December 30, 2019. No specific pathogens (including HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1) were detected in clinical specimens from these patients by the RespiFinderSmart22kit. RNA extracted from bronchoalveolar-lavage fluid from the patients was used as a template to clone and sequence a genome using a combination of Illumina sequencing and nanopore sequencing. More than 20,000 viral reads from individual specimens were obtained, and most contigs matched to the genome from lineage B of the genus betacoronavirus — showing more than 85% identity with a bat SARS-like CoV (bat-SL-CoVZC45, MG772933.1) genome published previously. Positive results were also obtained with use of a real-time RT-PCR assay for RNA targeting to a consensus RdRp region of pan β-CoV (although the cycle threshold value was higher than 34 for detected samples). Virus isolation from the clinical specimens was performed with human airway epithelial cells and Vero E6 and Huh-7 cell lines. The isolated virus was named 2019-nCoV.”

“Electron micrographs of negative-stained 2019-nCoV particles were generally spherical with some pleomorphism (Figure 3). Diameter varied from about 60 to 140 nm. Virus particles had quite distinctive spikes, about 9 to 12 nm, and gave virions the appearance of a solar corona. Extracellular free virus particles and inclusion bodies filled with virus particles in membrane-bound vesicles in cytoplasm were found in the human airway epithelial ultrathin sections. This observed morphology is consistent with the Coronaviridae family.”

Discussion

“We report a novel CoV (2019-nCoV) that was identified in hospitalized patients in Wuhan, China, in December 2019 and January 2020. Evidence for the presence of this virus includes identification in bronchoalveolar-lavage fluid in three patients by whole-genome sequencing, direct PCR, and culture. The illness likely to have been caused by this CoV was named “novel coronavirus-infected pneumonia” (NCIP). Complete genomes were submitted to GISAID. Phylogenetic analysis revealed that 2019-nCoV falls into the genus betacoronavirus, which includes coronaviruses (SARS-CoV, bat SARS-like CoV, and others) discovered in humans, bats, and other wild animals.15 We report isolation of the virus and the initial description of its specific cytopathic effects and morphology.

Molecular techniques have been used successfully to identify infectious agents for many years. Unbiased, high-throughput sequencing is a powerful tool for the discovery of pathogens.14,16 Next-generation sequencing and bioinformatics are changing the way we can respond to infectious disease outbreaks, improving our understanding of disease occurrence and transmission, accelerating the identification of pathogens, and promoting data sharing. We describe in this report the use of molecular techniques and unbiased DNA sequencing to discover a novel betacoronavirus that is likely to have been the cause of severe pneumonia in three patients in Wuhan, China.

Although establishing human airway epithelial cell cultures is labor intensive, they appear to be a valuable research tool for analysis of human respiratory pathogens.13 Our study showed that initial propagation of human respiratory secretions onto human airway epithelial cell cultures, followed by transmission electron microscopy and whole genome sequencing of culture supernatant, was successfully used for visualization and detection of new human coronavirus that can possibly elude identification by traditional approaches.”

Although our study does not fulfill Koch’s postulates, our analyses provide evidence implicating 2019-nCoV in the Wuhan outbreak. Additional evidence to confirm the etiologic significance of 2019-nCoV in the Wuhan outbreak include identification of a 2019-nCoV antigen in the lung tissue of patients by immunohistochemical analysis, detection of IgM and IgG antiviral antibodies in the serum samples from a patient at two time points to demonstrate seroconversion, and animal (monkey) experiments to provide evidence of pathogenicity. Of critical importance are epidemiologic investigations to characterize transmission modes, reproduction interval, and clinical spectrum resulting from infection to inform and refine strategies that can prevent, control, and stop the spread of 2019-nCoV.”

https://www.nejm.org/doi/full/10.1056/nejmoa2001017

Like Zhou et al. before them, Zhu et al. couldn’t satisfy these essential criteria to prove their “virus” exists and causes disease.

In Summary:

  • Zhu et al. collected samples, including BALF fluid, from 4 patients with pneumonia for whom they could not determine a cause
  • They used samples from 7 patients with pneumonia of known causes as “controls”
  • The BALF from the 7 patients was added to Viral Transport Medium
  • They used human airway epithelial cells from lung cancer patients to culture their “virus” which were regularly washed with and stored in phosphate-buffered saline which can be toxic to cells:

Viral Transport Media

  • The unpurified cell culture supernatant was used for the EM images in the study
  • On top of the numerous toxic chemicals used during the culturing/washing process, paraformaldehyde was added to the supernatant for 2 hours to prepare it for TEM imaging
  • More paraformaldehyde as well as glutaraldehyde were added and then the sample was fixed with osmium tetroxide dehydrated with grade ethanol and embedded in resin
  • Sections were cut from the resin and stained with uranyl acetate and lead citrate
  • These TEM preparation processes not only kill and alter the cells, they can create artifacts in the TEM images as well:

Electron Microscope Imagery: “Virus” Proof or Refutation?

The Numerous Alterations During Sample Preparation for EM Imaging

  • RNA extracted from the unpurified BALF and culture supernatant was used as a template to clone and generate the genome
  • The genome shared more than 85% identity match to the bat “Coronavirus” RaTG13
  • PCR Ct Values were higher than 34 for the detected samples (which, according to Fauci, would be nothing but dead nucleotides)
  • “Virus” isolation was carried out in human airway epithelial cells, Vero cells, and HuH7 cells
  • They observed “Coronavirus-like’ particles with some pleomorphism (variability of size, shape, and staining of cells) in their unpurified cell culture sample
  • Their evidence consists of whole-genome sequencing from unpurified BALF of 3 patients, direct PCR, and cell culture
  • The illness likely to have been caused by their new “virus” was named 2019-nCoV
  • Zhu et al. talk up their indirect molecular techniques as being suitable to identify a novel “virus” which is likely to be the cause of an unknown pneumonia
  • They claim the “isolation” of the “virus” and the initial description of its specific cytopathic effects and morphology yet without purification, this is an outright lie
  • They state that they were able to capture and identify this unknown “virus” through TEM images, cell cultures, and WGS sequencing from unpurified cell culture supernatant as it may have eluded identification by traditional approaches
  • The researchers then admit that they did not satisfy Koch’s Postulates, the very criteria needed to be met in order to prove a new pathogenic “virus” exists
  • They also admit more evidence is needed to confirm its etiological significance such as:
    1. Identifying an antigen
    2. Detection of IgG and IgM antibodies at two time intervals to show seroconversion
    3. Animal experiments to prove pathogenicity
  • Zhu et al. concludes that further studies are also needed to:
    1. Characterize transmission modes
    2. Reproduction intervals
    3. Determine clinical spectrum

Once you break down the “evidence” (or lack thereof), it is clear to see that the world was locked down, quarantined, masked, social distanced, and vaccinated based on nothing at all. Neither the Zhu nor the Zhou studies fulfilled Koch’s postulates. Both left it up to future studies from different teams of researchers using different patients with different samples and different methods to prove their hypotheses for them. At the very least, Zhu provided a pretty TEM image of some particles he picked to represent his “virus” from potentially billions of similar particles in the unpurified cell culture supernatant.

“SARS-COV-2?”
Specific CPE?

However, TEM images of particles that may or may not belong to the A’s, C’s, G’s, and T’s in a computer database that may or may not represent something in reality is not evidence of a new “virus.” It is absolute fraud to present it as such.

For more on TEM images and why purification/isolation of the particles is absolutely essential, read these related posts:

The Mimickers of “SARS-COV-2”

Misinterpreting Electron Microscope Images

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