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.

9 comments

  1. Have you read the Lanka 3-part interview with DSalud? (it’s in the files) In part 2, under about the 7th question (I don’t have a page number), The question was about the primers, and how they use as primers genetic fragments that are present in hundreds of microbes….. if so, why don’t they all test positive?” LANKA: Before the Chinese and other researchers created these genomes, they removed all known sequences. What happens here is that long sequences are discarded that the computer detects as being from microbes but smaller fragments are found in the viral genome they build. This is obviously not discussed. I repeat: all known sequences that are available on the internet are removed from the set of what has been sequenced and only then alignment begins. This is why so few actual known sequences are found in the virus genome and only those that were unknown at the time of creation are found. The known ones cannot be found because they were filtered out and what later appear are only a few fragments since the length of their sequencing is only 150
    nucleotides. Large pieces of microbial genetic information never appear due to the technology used which carries out a conceptual mathematical sequencing of the virus genome.”
    Okay, so they purposely use short fragments because the long ones are of microorganisms that they already “know”? So is there way of “isolating” — by just getting rid of the “stuff in the soup that is not a virus” ?? And they are assuming that the short fragments are “new” and “novel” and must be a virus?????
    I think I might be confused about the primers and the current test they are doing… like which comes first….. but in general, do I have this right?
    Maybe I already knew this…. this is the in silico process of creating a genome from fragments. But this part that always happens before they even start alignment (that is the part where they fill the “holes” in the sequence in, right?) is where they MENTALLY AND THROUGH SOFTWARE “isolate” the genetic fragments that they ASSUME are virus fragments?
    When really they made a soup, took out all the genetic sequences that were “carrot” and “potato” and then whatever was left over (the broken down stuff) is called a virus????

    Liked by 1 person

    1. I have not read that yet but that is a great explanation. As far as my understanding goes, you are correct. They assume what is left comes from one source yet we know for a fact not all sequences are known and that off-target genetic material is always present. We also know numerous other particles like extracellular vesicles will be present. They are just taking random puzzle pieces and trying to get them to fit whatever mold they choose using computer algorithms and prediction software. Nothing but a computerized fantasy.

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      1. It really feels like one of the main tricks they do (self deception?) is in this step that Lanka describes in that quote….. where they eliminate the “other stuff in the soup” through the software. Do you think in a way this is the “isolation” step??? Like a mental filtering and purifying that occurs in the software and In their minds. They isolate and CREATE a new “thing” in their minds. I like when Lanka says, “they don’t discuss this”. uh, yeah! Not that anyone cares anyway. They could do just about anything and if they train the doctors, lab techs, scientists in a way where it all seems justified and real, then it IS real in a sense. But it’s like symbolically real, not really real. That’s the trouble, the map (symbols) are not the territory (the body, nature, reality as it really is). They are playing with symbols and THAT has been justified in our minds, especially with viruses, because they are invisible! Kind of like a symbol itself. It’s not really there, it’s just an idea. So they feel they can play with it. But what is amazing is when you talk to doctors who have taken biochemistry and virology, they teach them in a way where it seems real, not symbolic. I just had a long conversation about all this yesterday with an infectious disease doctor. WOW. She talked about how she had a lab where she had to find the viruses in thee professor’s infant son’s poop! And she has used the PCR machine and it works and is “only as good as the primers”, meaning any fault with using the machine is because the primers aren’t good enough…. well yeah! how viruses eat bacteria and are essential for life etc… it’s all very real to her, not at all symbolic. And how antivirals work… it’s very hard to explain the “take down” of virology that is being uncovered. She thinks Covid is real, that people are dying now from “misinformation”. She has NOT encountered the real information though. Lanka etc. That is the key. To present it to her in a way that is VERY solid. It’s a long story how I know her (my own health backstory) but she is very bright and is actually quite rebellious by nature (she is featured in a film on Morgellen’s, she treats people who have been told they are not sick, that they are crazy…..). So she thinks it is ironic that now she is defending the CDC which she has been battling in the past. It’s all very complex. LIke a tangled ball of yarn. Sorry this got so long.

        Liked by 1 person

      2. Yes, it is nothing but a mental (or computerized) construct built on the assumption that all sources of the “other stuff” in the soup are known and accurate. They will try to claim this is purification (had a long circular “debate” with someone about this) but it is not at all. Purification means free of any foreign material, contaminants, pollutants, etc. The very fact that there are numerous sources of off-tarfet genetic material in the sample proves no purification/isolation ever took place. They can never be certain the genetic material is not coming from multiple sources which are then being put together in a fictional mold. In fact, it is very apparent this is the case.

        As for your friend, all we can really do is point this information out. If she is open-minded and has the ability to think critically/logically, she will eventually see the fallacies. It just might not happen until she finds the proper internal motivator.

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      3. so people really do claim that the software is doing the isolating??? The thing is, the public has no idea this is what they are doing. They think they have “got” the virus, then they have genetically analyzed it. They don’t get that the cell culturing is MAKING AN EFFECT!! And that effect is seen as it “must” be the effect of the virus! Yet it is CLEARLY the effect of the culturing process!!! With no controls! So that story is what people don’t get… I say story because it needs to be told as a story, people don’t know what is happening because it needs to be told in a way they understand.

        Liked by 1 person

      4. Absolutely. This is why I put so much effort into breaking down the cell culture method. Once people truly understand it, there is no way to believe it is valid evidence nor would any genome coming from it.

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      5. This is so true with all the things that we are finding out are just not true, not scientific, not logical, and have no evidence. If people were walked through the science on so many topics… so much is revealed. So, I guess that is what we are “fighting” — the addiction to fantasy, the lack of being able to track the science, even by scientists!!!! It’s why that video freaked me out, the cartoon one, because it was all fantasy and the comments are all like, “yeah, that is reality!”

        Liked by 1 person

      6. yes, it is possible and if we imagine it happening (spell broken!) it will happen….. and, if you really want to get deep and woo woo about it, it already HAS happened! Because I believe that humanity has decided to wake up. I do. It’s just taking a while because us humans like to sleep in, ha!

        Liked by 1 person

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