Zhou “SARS-COV-2” Paper (2020)

It is clear after having gone through the history of “Coronaviruses” from 1965 up to today, not a single one of these so-called “viruses” has ever been properly purified/isolated directly from a sick patient nor proven pathogenic by fulfilling Koch’s Postulates. They always take the fluid from a sick patient and mix it with animal cells (usually from an African Green Monkey Kidney called Vero cells) along with a combination of antibiotics/antifungals, fetal bovine serum, “nutrients,” and other chemicals. Even from this concoction, which can hardly be called an isolation of anything, they never purify any “virus” particles. Sometimes they take EM images directly from the cell culture supernatant which contains potentially billions of similar looking particles. They never prove pathogenicity in a natural way in animal models. This is as true today with “SARS-COV-2” as it was in 1965 with the forgotten B814. Presented below are highlights from one of the first “SARS-COV-2” studies from February 2020:

A pneumonia outbreak associated with a new coronavirus of probable bat origin

“Here we report the identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started on 12 December 2019, had caused 2,794 laboratory-confirmed infections including 80 deaths by 26 January 2020. Full-length genome sequences were obtained from five patients at an early stage of the outbreak. The sequences are almost identical and share 79.6% sequence identity to SARS-CoV. Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus.”

“The disease was determined to be caused by virus-induced pneumonia by clinicians according to clinical symptoms and other criteria, including a rise in body temperature, decreases in the number of lymphocytes and white blood cells (although levels of the latter were sometimes normal), new pulmonary infiltrates on chest radiography and no obvious improvement after treatment with antibiotics for three days.”

“Samples from seven patients with severe pneumonia (six of whom are sellers or deliverymen from the seafood market), who were admitted to the intensive care unit of Wuhan Jin Yin-Tan Hospital at the beginning of the outbreak, were sent to the laboratory at the Wuhan Institute of Virology (WIV) for the diagnosis of the causative pathogen (Extended Data Table 1). As a laboratory investigating CoV, we first used pan-CoV PCR primers to test these samples¹³, given that the outbreak occurred in winter and in a market—the same environment as SARS infections. We found five samples to be PCR-positive for CoVs. One sample (WIV04), collected from the bronchoalveolar lavage fluid (BALF), was analysed by metagenomics analysis using next-generation sequencing to identify potential aetiological agents. Of the 10,038,758 total reads—of which 1,582 total reads were retained after filtering of reads from the human genome—1,378 (87.1%) sequences matched the sequence of SARSr-CoV (Fig. 1a). By de novo assembly and targeted PCR, we obtained a 29,891-base-pair CoV genome that shared 79.6% sequence identity to SARS-CoV BJ01 (GenBank accession number AY278488.2). High genome coverage was obtained by remapping the total reads to this genome (Extended Data Fig. 1).”

“We then found that a short region of RNA-dependent RNA polymerase (RdRp) from a bat coronavirus (BatCoV RaTG13)—which was previously detected in Rhinolophus affinis from Yunnan province—showed high sequence identity to 2019-nCoV. We carried out full-length sequencing on this RNA sample (GISAID accession number EPI_ISL_402131). Simplot analysis showed that 2019-nCoV was highly similar throughout the genome to RaTG13 (Fig. 1c), with an overall genome sequence identity of 96.2%. Using the aligned genome sequences of 2019-nCoV, RaTG13, SARS-CoV and previously reported bat SARSr-CoVs, no evidence for recombination events was detected in the genome of 2019-nCoV. Phylogenetic analysis of the full-length genome and the gene sequences of RdRp and spike (S) showed that—for all sequences—RaTG13 is the closest relative of 2019-nCoV and they form a distinct lineage from other SARSr-CoVs (Fig. 1d and Extended Data Fig. 2). The receptor-binding spike protein encoded by the S gene was highly divergent from other CoVs (Extended Data Fig. 2), with less than 75% nucleotide sequence identity to all previously described SARSr-CoVs, except for a 93.1% nucleotide identity to RaTG13 (Extended Data Table 3). The S genes of 2019-nCoV and RaTG13 are longer than other SARSr-CoVs.”

“The close phylogenetic relationship to RaTG13 provides evidence that 2019-nCoV may have originated in bats.”

“We rapidly developed a qPCR-based detection method on the basis of the sequence of the receptor-binding domain of the S gene, which was the most variable region of the genome (Fig. 1c). Our data show that the primers could differentiate 2019-nCoV from all other human coronaviruses including bat SARSr-CoV WIV1, which shares 95% identity with SARS-CoV (Extended Data Fig. 4a, b). Of the samples obtained from the seven patients, we found that six BALF and five oral swab samples were positive for 2019-nCoV during the first sampling, as assessed by qPCR and conventional PCR. However, we could no longer detect virus-positive samples in oral swabs, anal swabs and blood samples taken from these patients during the second sampling (Fig. 2a). However, we recommend that other qPCR targets, including the RdRp or envelope (E) genes are used for the routine detection of 2019-nCoV. On the basis of these findings, we propose that the disease could be transmitted by airborne transmission, although we cannot rule out other possible routes of transmission, as further investigation, including more patients, is required.”

“We next successfully isolated the virus (called 2019-nCoV BetaCoV/Wuhan/WIV04/2019) from both Vero E6 and Huh7 cells using the BALF sample of patient ICU-06. Clear cytopathogenic effects were observed in cells after incubation for three days (Extended Data Fig. 6a, b). The identity of the strain WIV04 was verified in Vero E6 cells by immunofluorescence microscopy using the cross-reactive viral N antibody (Extended Data Fig. 6c, d) and by metagenomics sequencing, most of the reads of which mapped to 2019-nCoV, and qPCR analysis showed that the viral load increased from day 1 to day 3 (Extended Data Fig. 6e, f). Viral particles in ultrathin sections of infected cells displayed a typical coronavirus morphology, as visualized by electron microscopy (Extended Data Fig. 6g).”

“The study provides a detailed report on 2019-nCoV, the likely aetiological agent responsible for the ongoing epidemic of acute respiratory syndrome in China and other countries. Virus-specific nucleotide-positive and viral-protein seroconversion was observed in all patients tested and provides evidence of an association between the disease and the presence of this virus. However, there are still many urgent questions that remain to be answered. The association between 2019-nCoV and the disease has not been verified by animal experiments to fulfil the Koch’s postulates to establish a causative relationship between a microorganism and a disease. We do not yet know the transmission routine of this virus among hosts.”

Sample collection

“Human samples, including oral swabs, anal swabs, blood and BALF samples were collected by Jinyintan hospital (Wuhan, China) with the consent of all patients and approved by the ethics committee of the designated hospital for emerging infectious diseases. Patients were sampled without gender or age preference unless indicated. For swabs, 1.5 ml DMEM containing 2% FBS was added to each tube. The supernatant was collected after centrifugation at 2,500 rpm, vortexing for 60 s and a standing period of 15–30 min. The supernatant from swabs or BALF (no pre-treatment) was added to either lysis buffer for RNA extraction or to viral transport medium for isolation of the virus. The viral transport medium was composed of Hank’s balanced salt solution (pH 7.4) containing BSA (1%), amphotericin (15 μg ml⁻¹), penicillin G (100 units ml⁻¹) and streptomycin (50 μg ml⁻¹). Serum was separated by centrifugation at 3,000g for 15 min within 24 h of collection, followed by inactivation at 56 °C for 1 h, and was then stored at 4 °C until use.

Virus isolation, cell infection, electron microscopy and neutralization assay

The following cell lines were used for virus isolation in this study: Vero E6 and Huh7 cells, which were cultured in DMEM containing 10% FBS. All cell lines were tested and free of mycoplasma contamination, submitted for species identification and authenticated by morphological evaluation by microscopy. None of the cell lines was on the list of commonly misidentified cell lines (by ICLAC).

Cultured cell monolayers were maintained in their respective medium. The PCR-positive BALF sample from ICU-06 patient was spun at 8,000g for 15 min, filtered and diluted 1:2 with DMEM supplemented with 16 μg ml⁻¹ trypsin before it was added to the cells. After incubation at 37 °C for 1 h, the inoculum was removed and replaced with fresh culture medium containing antibiotics (see below) and 16 μg ml⁻¹ trypsin. The cells were incubated at 37 °C and observed daily for cytopathogenic effects. The culture supernatant was examined for the presence of virus by qRT–PCR methods developed in this study, and cells were examined by immunofluorescence microscopy using the anti-SARSr-CoV Rp3 N antibody that was generated in-house (1:1,000). Penicillin (100 units ml⁻¹) and streptomycin (15 μg ml⁻¹) were included in all tissue culture media.

Vero E6 cells were infected with the new virus at a multiplicity of infection (MOI) of 0.5 and collected 48 h after infection. Cells were fixed with 2.5% (w/v) glutaraldehyde and 1% osmium tetroxide, dehydrated through a graded series of ethanol concentrations (from 30 to 100%) and embedded with epoxy resin. Ultrathin sections (80 nm) of embedded cells were prepared, deposited onto Formvar-coated copper grids (200 mesh), stained with uranyl acetate and lead citrate, and analysed using a 200-kV Tecnai G2 electron microscope.

The virus neutralization test was carried out in a 96-well plate. The patient serum samples were heat-inactivated by incubation at 56 °C for 1 h before use. The serum samples were diluted to 1:10, 1:20, 1:40 or 1:80, and then an equal volume of virus stock was added and incubated at 37 °C for 60 min in a 5% CO₂ incubator. Diluted horse anti-SARS-CoV serum or serum samples from healthy individuals were used as control. After incubation, 100 μl mixtures were inoculated onto a monolayer of Vero E6 cells in a 96-well plate for 1 h. Each serum was assessed in triplicate. After removing the supernatant, the plate was washed twice with DMEM medium. Cells were incubated with DMEM supplemented with 2% FBS for 3 days. Subsequently, the cells were checked for cytopathogenic effects.”

Examination of ACE2 receptor for 2019-nCoV infection

“HeLa cells transiently expressing ACE2 were prepared using Lipofectamine 3000 (Thermo Fisher Scientific) in a 96-well plate; mock-transfected cells were used as controls. 2019-nCoV grown in Vero E6 cells was used for infection at a MOI of 0.5. APN and DPP4 were analysed in the same way. The inoculum was removed after absorption for 1 h and washed twice with PBS and supplemented with medium. At 24 h after infection, cells were washed with PBS and fixed with 4% formaldehyde in PBS (pH 7.4) for 20 min at room temperature. ACE2 expression was detected using a mouse anti-S tag monoclonal antibody and a FITC-labelled goat anti-mouse IgG H&L (Abcam, ab96879). Viral replication was detected using a rabbit antibody against the Rp3 N protein (generated in-house, 1:1,000) and a Cy3-conjugated goat anti-rabbit IgG (1:200, Abcam, ab6939). Nuclei were stained with DAPI (Beyotime). Staining patterns were examined using confocal microscopy on a FV1200 microscope (Olympus).

High-throughput sequencing, pathogen screening and genome assembly

Samples from patient BALF or from the supernatant of virus cultures were used for RNA extraction and next-generation sequencing (NGS) using BGI MGISEQ2000 and Illumina MiSeq 3000 sequencers.”

“A local nucleic acid database for human and mammals was used to filter reads of host genomes before mapping reads to the virus database. The results of the metagenomic analysis were displayed as pie charts using Microsoft Office 2010. NGS reads were assembled into genomes using Geneious (v.11.0.3) and MEGAHIT (v.1.2.9). PCR and Sanger sequencing was performed to fill gaps in the genome. 5′-rapid amplification of cDNA ends (RACE) was performed to determine the 5′-end of the genomes using a SMARTer RACE 5′/3′ kit (Takara). Genomes were annotated using the Clone Manager Professional Suite 8 (Sci-Ed Software).”

https://www.nature.com/articles/s41586-020-2012-7#ref-CR13

In Summary:

• The discovery of this new “Coronavirus” started from the sequencing of a genome from the unpurified BALF of sick patients
• The sequence only had a 79.8% sequence match to the original SARS
• The “SARS-COV-2” sequence, however, was shown to be 96.2% similar to a bat “Coronavirus” named RaTG13
• The cases of disease were determined to be caused by a “virus” based on clinical symptoms and other measures as well as the lack of improvement after 3 days of antibiotic use
• The researchers tested samples from seven patients with “Coronavirus” PCR primers based on the hunch that it may be “SARS” due to the geographical location of the patients
• For swabs, 1.5 ml DMEM containing 2% Fetal Bovine Serum was added to each tube
• 5 of the 7 tested positive by PCR for “Coronaviruses” and the BALF from one patient was sent for metagenomic sequencing as detailed here:

Creating the “SARS-COV-2” Genome

• They utilized the highly questionable “SARS-COV-1” as the reference genome to remap the reads to get the “SARS-COV-2” genome
• They found that the new “virus” was most closely related (96.2%) to the also highly questionable bat “Coromavirus” RaTG13:

“SARS-COV-2” and the Slippery Slope of Reference Genomics

The Mysterious Animal Origin of “SARS-COV-2” Part 1: Bats

• Next, the team quickly developed their own PCR test to detect “SARS-COV-2” in the BALF of 6 out of 7 patients and from the oral swabs of 5 out of 7 patients
• It was finally decided to “isolate” the “virus” in Vero and HuH7 cell cultures from the BALF sample from one patient AFTER they had determined their genome and made their own PCR test
• To understand the numerous effects the additives discussed in this next section have on the cell culture process and how this can create the effect virologists look for, see this link:

The Case Against Cell Cultures

• For “isolation,” the swabs were placed in viral transport medium composed of Hank’s balanced salt solution (pH 7.4) containing BSA, amphotericin, penicillin G, and streptomycin
• Vero E6 (from African Green Monkey kidneys) and Huh7 cells (a cancerous cell line taken from the liver of a 57-year-old Japanese man) were cultured in DMEM containing 10% FBS
• The PCR-positive BALF sample from ICU-06 patient was spun at 8,000g for 15 min, filtered and diluted 1:2 with DMEM supplemented with trypsin before it was added to the cells
• After incubation at 37 °C for 1 h, the inoculum was removed and replaced with fresh culture medium containing antibiotics and trypsin
• Penicillin and streptomycin, which are toxic to cells and can cause the cytopathogenic effect (CPE) they look for as proof of ‘virus,” were added to all tissue cultures
• They next looked at unpurified cell culture supernatant in an Electron Microscope
• Cells were fixed with 2.5% (w/v) glutaraldehyde and 1% osmium tetroxide, dehydrated through a graded series of ethanol concentrations (from 30 to 100%) and embedded with epoxy resin
• Ultrathin sections (80 nm) of embedded cells were prepared, deposited onto Formvar-coated copper grids (200 mesh), stained with uranyl acetate and lead citrate, and analysed using a 200-kV Tecnai G2 electron microscope
• They saw “Coronavirus-like” particles (from which there are many similar looking particles within the sample) and decided that this was their “virus”
• After doing “neutralization” tests using the predetermined diluted horse anti-SARS-CoV serum, 100 μl mixtures were inoculated onto a monolayer of Vero E6 cells in a 96-well plate for 1 h
• They removed the supernatant and the plate was washed twice with DMEM medium and the cells were incubated with DMEM supplemented with 2% FBS for 3 days
• After the many cell-altering toxins were added to the cells, they were checked for the indirect and nonspecific evidence of cytopathogenic effects to conclude a “virus” was in the sample
• It was concluded that “SARS-COV-2” is the LIKELY aetiological agent causing disease
• The team then admit that they did not fulfill Koch’s Postulates to actually determine whether or not their new “virus” actually causes disease
• They also admit that animal studies were still needed in order to see if they could reproduce the same disease as seen in humans
• The mode of transmission for the “virus” was unknown

As with MERS before it, this whole study and the hysteria surrounding “SARS-COV-2” can be completely thrown out due to the researchers admitting that they never fulfilled Koch’s Postulates nor proved that their letters in a database actually exists nor causes disease. They mention EM images yet never supplied any in the study. They never mention any attempts at purification. They started with a genome before they ever attempted “isolating” a “virus.” The genomes used as references to create “SARS-COV-2” came from unpurified and highly questionable sources. This paper is one big fraudulent mess.

If you can not see the lies of virology by reading the original papers supplied as evidence, you aren’t trying very hard as they are all there, clear as day for those who are willing to see.