Are Viruses Real?

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Mashing up colloquial uses of the term doesn't change the definition used within the sciences. A theory is not a hypothesis. It is not a belief or conjecture. These are popular, but not scientific uses of the word. When someone is talking about gravitational theory, or evolutionary theory, or germ theory, they aren't talking about "just a theory" in the popular sense you're trying to imply, but in the scientific sense of a set of rigourously tested, replicable, predictive statements and rules.

Which is the entire point to my saying I do not believe in something which hasn't been tested
The fact that you reject the tests and proofs doesn't make them magically stop existing.
 
Mashing up colloquial uses if the term doesn't change the definition used within the sciences. A theory is not a hypothesis. It is not a belief or conjecture. These are popular, but not scientific uses if the word. When someone is talking about gravitational theory, or evolutionary theory, they aren't talking about "just a theory" in the popular sense you're trying to imply, but in the scientific sense of a set of rigourously tested, replicable, predictive statements and rules.
Cool anecdotes, guy. Unfortunately, not only are you up against the dictionary but you're also going to have to reinvent the etymology of the words too. Good luck with that. 😜

The fact that you reject the tests and proofs doesn't make them magically stop existing.
This is another strawman. I never rejected any tests or proofs. I simply acknowledged the meaning of words.

I recommend exploring a cornfield and be a faggot to an actual scarecrow if this is what you enjoy.
 
Cool anecdotes, guy. Unfortunately, not only are you up against the dictionary
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The Difference Between Hypothesis and Theory

A hypothesis is an assumption, an idea that is proposed for the sake of argument so that it can be tested to see if it might be true.
In the scientific method, the hypothesis is constructed before any applicable research has been done, apart from a basic background review. You ask a question, read up on what has been studied before, and then form a hypothesis.
A hypothesis is usually tentative; it's an assumption or suggestion made strictly for the objective of being tested.
A theory, in contrast, is a principle that has been formed as an attempt to explain things that have already been substantiated by data. It is used in the names of a number of principles accepted in the scientific community, such as the Big Bang Theory. Because of the rigors of experimentation and control, it is understood to be more likely to be true than a hypothesis is.
In non-scientific use, however, hypothesis and theory are often used interchangeably to mean simply an idea, speculation, or hunch, with theory being the more common choice.
Since this casual use does away with the distinctions upheld by the scientific community, hypothesis and theory are prone to being wrongly interpreted even when they are encountered in scientific contexts—or at least, contexts that allude to scientific study without making the critical distinction that scientists employ when weighing hypotheses and theories.
The most common occurrence is when theory is interpreted—and sometimes even gleefully seized upon—to mean something having less truth value than other scientific principles. (The word law applies to principles so firmly established that they are almost never questioned, such as the law of gravity.)
This mistake is one of projection: since we use theory in general to mean something lightly speculated, then it's implied that scientists must be talking about the same level of uncertainty when they use theory to refer to their well-tested and reasoned principles.
The distinction has come to the forefront particularly on occasions when the content of science curricula in schools has been challenged—notably, when a school board in Georgia put stickers on textbooks stating that evolution was "a theory, not a fact, regarding the origin of living things." As Kenneth R. Miller, a cell biologist at Brown University, has said, a theory "doesn’t mean a hunch or a guess. A theory is a system of explanations that ties together a whole bunch of facts. It not only explains those facts, but predicts what you ought to find from other observations and experiments.”
While theories are never completely infallible, they form the basis of scientific reasoning because, as Miller said "to the best of our ability, we’ve tested them, and they’ve held up."

Two Related, Yet Distinct, Meanings of Theory


There are many shades of meaning to the word theory. Most of these are used without difficulty, and we understand, based on the context in which they are found, what the intended meaning is. For instance, when we speak of music theory we understand it to be in reference to the underlying principles of the composition of music, and not in reference to some speculation about those principles.


However, there are two senses of theory which are sometimes troublesome. These are the senses which are defined as “a plausible or scientifically acceptable general principle or body of principles offered to explain phenomena” and “an unproven assumption; conjecture.” The second of these is occasionally misapplied in cases where the former is meant, as when a particular scientific theory is derided as "just a theory," implying that it is no more than speculation or conjecture. One may certainly disagree with scientists regarding their theories, but it is an inaccurate interpretation of language to regard their use of the word as implying a tentative hypothesis; the scientific use of theory is quite different than the speculative use of the word

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You are an ignorant faggot.
 
You are an ignorant faggot.

I still see absolutely nothing that dissuades from anything I said. In fact, the first screenshot specifically talks about the word "law" which is not the same as "theory." Also, keen to note that what is mentioned is these are almost never questioned. Meaning they are questioned, but not too often. Which is appropriate since science never settles on any singular answer.

Second screenshot says "ideas" and "suggested" which means the language is still utterly and completely speculative. This is why they've been appropriately termed "theories" as true to the etymonology. So, both your "proofs" are in my favor. Sorry, but not sorry.

Words mean things regardless of what you want them to mean, faggot.
 
@Cheeseknife is one weird motherfucker. With every post he exhibits more and more of the behaviors he previously attributed to other people. I don't know if this is some kind of attempt to turn the tables against the people arguing with him by behaving like the wilfully ignorant strawman he's constructed of others, or if his entire personality is built upon psychological projection. Either way, as a case study he's almost as fascinating as Doctor Bailey's paper.
Almost.
Top Gear meme tax:
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Wiles also decided to champion virology’s blatant misuse of the word ‘isolation’ when she stated, “as for using isolation in the every-day sense of the word, rather than the definition that is relevant to the question being asked? Well, that’s just bloody ridiculous and a clear sign these requests for evidence are not being made in good faith.” She appeared to be incredulous that others had pointed out that the definition of a word being used scientifically was unilaterally changed by the virologists to imply a certain proof was obtained. However, if their use of isolation does not mean what most people think it means, then it is likely that most of the public are being misinformed. On this account, Wiles is an active participant in promulgating disinformation, whether it is an act of wilful blindness or otherwise. Wiles needs to show her hand as an expert and explain to the
public what the definition of isolation in virology means, in particular with regard to demonstrating the putative existence of viruses. Perhaps she thinks she did explain when she wrote, “when virologists want to isolate a virus from a sample they’ll take the sample or some part of it and add it to some cells – usually ones that are relatively easy to grow in the lab – and then look to see if the cells die and/or if there are any virus particles released into the liquid nutrient bath the cells are growing in.” It is unclear if Wiles is implying that the “virus isolate” is established by: (a) the
taking of the sample, (b) seeing some cells die in vitro, (c) the release of claimed “virus particles” in the tissue culture, or (d) all or some combination of these elements. However, nothing she described requires the existence of viruses — it is a game of deception, whether realised or not. It
simply involves the assertion that a virus was in the sample, blaming the breakdown of experimentally stressed cells in the test tube on the imagined virus, and then declaring that some of the vesicles (whose biological composition and function were not established) were the viruses.
There is a further fatal flaw in this exercise. As this essay will detail, the claims that SARS-CoV-2 has been shown to exist through this methodology are all scientifically invalid as none of the experiments were performed with valid controls.

This is exemplary of how Wiles has acted in her role as one of the key influencers for the New Zealand government’s disinformation campaign and its murderous rollout programme of an injectable product called Comirnaty™ – claiming that non-specific tissue culture experiments verify the existence of the virus when nothing of the kind has been demonstrated. The issue extends beyond just SARS-CoV-2 — every virus asserted to exist relies on similar pseudoscience. The history of virology reveals that the types of cells eventually selected for these experiments have been those that have a propensity to breakdown with the claim of virus-induced ‘cytopathic effects’ (CPEs), rather than those that are, “relatively easy to grow in the lab,” as Wiles claimed in her article. For example, Vero E6 monkey cells have long been favoured by virologists, supposedly due to their “suitability” to host many viruses, but suspiciously also, because the aneuploid kidney line is more susceptible to toxic insults from additional ingredients such as the ubiquitous nephrotoxic antibiotics and antifungals added to the culture mix. When one group attempted to culture SARS-CoV-2, they had no desired result with human adenocarcinoma cells (A549), human liver cells (HUH7.0), human embryonic kidney cells (HEK-293T), and a big brown bat kidney cell line (EFK3B), but then declared they had a “viral isolate” following the observation of CPEs in Vero E6 cells. As is typical, there seemed to be no sense of irony for them that the purported human respiratory virus cannot be shown to “infect” the relevant cell type, let alone the relevant species. And their experiments were once again invalidated by the absence of appropriate control cultures.
Last time on Faggot Ball Z:
Wiles also decided to champion virology’s blatant misuse of the word ‘isolation’ when she stated, “as for using isolation in the every-day sense of the word, rather than the definition that is relevant to the question being asked? Well, that’s just bloody ridiculous and a clear sign these requests for evidence are not being made in good faith.”
The more I read this the more I find myself agreeing with the dangerhair vaccine shill. What's the world coming to?
Based on what I've seen so far it seems like Bailey et al. are not arguing against virus theory from a place of good faith. Maybe for the others in their circle it's out of a genuine desire to advance science, but the Baileys themselves seem motivated by spite based on the frightful amount of high-inference language in this paper.
If I were a tremendous fag I'd say this taints their entire argument, but any rhetorician worth their salt knows that just because you commit a fallacy doesn't mean you're immediately wrong and your arguments aren't worth consideration. That assumption is a fallacy in and of itself.
Besides, we're still only on the eighth page. Let's soldier on.
She appeared to be incredulous that others had pointed out that the definition of a word being used scientifically was unilaterally changed by the virologists to imply a certain proof was obtained. However, if their use of isolation does not mean what most people think it means, then it is likely that most of the public are being misinformed. On this account, Wiles is an active participant in promulgating disinformation, whether it is an act of wilful blindness or otherwise. Wiles needs to show her hand as an expert and explain to the public what the definition of isolation in virology means, in particular with regard to demonstrating the putative existence of viruses.
Here we see more of Bailey's sensational language. The claim that the definition of "isolation" was changed to prove the existence of viruses is specious at this point as there has been no proof thus far that this is the case, at least as far as I can tell. Maybe one of my readers can point me in the direction of some proof while I'm waiting for Doctor Bailey to get around to it.
That being said, I agree that the scientific definition of "isolation" should be made publicly known, since it's apparent that it isn't the same as the colloquial definition. Maybe it has been somewhere and I just don't know about it.
Perhaps she thinks she did explain when she wrote, “when virologists want to isolate a virus from a sample they’ll take the sample or some part of it and add it to some cells – usually ones that are relatively easy to grow in the lab – and then look to see if the cells die and/or if there are any virus particles released into the liquid nutrient bath the cells are growing in.” It is unclear if Wiles is implying that the “virus isolate” is established by: (a) the
taking of the sample, (b) seeing some cells die in vitro, (c) the release of claimed “virus particles” in the Essue culture, or (d) all or some combination of these elements. However, nothing she described requires the existence of viruses — it is a game of deception, whether realised or not.
More loaded language. Like with Wiles refusing to cite a single source, I'm gonna assume the rest of the paper continues in this vein and stop harping on about it unless a particular phrase sticks out to me. Like "game of deception". How did that end up in a scientific paper? This is reading like the kind of journoshit I'd see in A&N more than any scientific essay.
I'd ask how nothing Wiles described required the existence of viruses but unlike with most claims made in this article, Bailey actually follows this up with what I assume is an explanation:
It simply involves the assertion that a virus was in the sample, blaming the breakdown of experimentally stressed cells in the test tube on the imagined virus, and then declaring that some of the vesicles (whose biological composition and function were not established) were the viruses.
This probably makes sense to people who actually know what's being talked about but I'm not part of that demographic. I'll leave it to you, the reader, to decide if that means anything to you.
There is a further fatal flaw in this exercise. As this essay will detail, the claims that SARS-CoV-2 has been shown to exist through this methodology are all scientifically invalid as none of the experiments were performed with valid controls.
"As this essay will detail"? Why not detail it now? Why waste time kvetching about some kiwi cunt with bad hair?
I acknowledge this is a nitpick but I feel like if I were in Doctor Bailey's position I wouldn't bury the lead like this. I may not know much about science, but I know a lot about persuasive essays. I feel like if I were interested in making a factual point I would damn persuasiveness and just exhibit the raw facts. I am, hypothetically, attempting to prove something which is based in fact; a violation of the scientific method. Exposing the alleged corruption of the scientific community (as I'm lead to believe is the point of this section) is the duty of a journalist, and taking on that duty as a scientist makes it seem as if I need to pad the facts to make them "truthier"—to borrow an expression from Stephen Cuckbear—for the reader. At the very least, I would make my biases known to others for the sake of transparency, which from my minimal understanding of the topic at hand is the bare minimum of a scientific paper.
Now I'm kvetching. On with the show.
This is exemplary of how Wiles has acted in her role as one of the key influencers for the New Zealand government’s disinformation campaign and its murderous rollout programme of an injectable product called Comirnaty™ – claiming that non-specific Essue culture experiments verify the existence of the virus when nothing of the kind has been demonstrated. The issue extends beyond just SARS-CoV-2 — every virus asserted to exist relies on similar pseudoscience.
More science gobbledygook, more loaded language. Much as I share in Bailey's skepticism of barely-tested vaccines I think describing the rollout as "murderous" is a little propagandistic. I am curious as to Bailey's explanation of the intent behind New Zealand's alleged disinformation campaign. I'm sure that'll be covered later, so that'll be exciting. As for "Essue culture experiments" I'll defer to people who know what the hell that means.

EDIT: It's not "Essue culture" but "tissue" culture. Copying directly from the PDF seems to render some letter combinations into single letters (ti=E, tt=g) and I guess I missed that one. I don't know why it does that in the first place.

I'm not sure if Comirnaty is an obscure word I'm not familiar with or if it's some corpo butchering of community but it sounds kind of silly.
The history of virology reveals that the types of cells eventually selected for these experiments have been those that have a propensity to breakdown with the claim of virus-induced ‘cytopathic effects’ (CPEs), rather than those that are, “relatively easy to grow in the lab,” as Wiles claimed in her article. For example, Vero E6 monkey cells have long been favoured by virologists, supposedly due to their “suitability” to host many viruses, but suspiciously also, because the aneuploid kidney line is more susceptible to toxic insults from additional ingredients such as the ubiquitous nephrotoxic antibiotics and antifungals added to the culture mix.
Why exactly is it suspicious? Is the argument here that the scientific community is obfuscating the true nature of viruses for some kind of gain or that the scientific community has been driven by collective confirmation bias to accept a bogus theory? While Bailey claims the former in his abstract, the evidence provided thus far seems to point to the latter. Not that the scientific community being objectively wrong for the past century or so is much better, but as of yet I fail to see the conspiracy beyond circumstantial evidence and emotive language on Bailey's part.
The citation in this passage is a link to a website where you can purchase the aforementioned Vero cells for $555.00 EA.
VERO C1008 [Vero 76, clone E6, Vero E6] is a cell line exhibiting epithelial morphology that was isolated from the kidney of an African green monkey. This line is a clone of VERO 76 (ATCC CRL-1587). It was cloned by the dilution method into microtiter plates in 1979 by P.J. Price. When infected with the hemorrhagic fever viruses [Machupo (Bolivian), Junin (Argentinian), Lassa (African)], Marburg or Ebola viruses, these cells exhibit cytopathic effects. Plaques are also produced. VERO C1008 exhibits some degree of contact inhibition after forming a monolayer and is therefore useful in growing slow replicating viruses. This cell line can be used in toxicology research.
Maybe here we see a piece of the potential motive for virology's alleged deception; the sale of expensive genetic material for the sake of lining some party's pockets. I don't know if that holds up, but it's something.
A footnote defines aneuploidy as:
the presence of an abnormal number of chromosomes in a cell.
Which is nice. I didn't know what that meant until I was told the definition. It begs the question why Bailey hasn't been defining all of his other terms. Maybe there's an appendix that lists all of the terms used. I'll see if I can find one later.
This is also completely unrelated but the use of the phrase "toxic insults" in this passage amuses me. I'm dimly aware of "insult" being a scientific term in this context, but it sounds like the cells used in these experiments are susceptible to being wounded by mean tweets.
Though now that I think about it, isn't "insult" also a word being used in a sense unrelated to its literal meaning? Why doesn't Bailey take issue with that? Maybe there's context I'm missing.
When one group agempted to culture SARS-CoV-2, they had no desired result with human adenocarcinoma cells (A549), human liver cells (HUH7.0), human embryonic kidney cells (HEK-293T), and a big brown bat kidney cell line (EFK3B), but then declared they had a “viral isolate” following the observation of CPEs in Vero E6 cells. As is typical, there seemed to be no sense of irony for them that the purported human respiratory virus cannot be shown to “infect” the relevant cell type, let alone the relevant species. And their experiments were once again invalidated by the absence of appropriate control cultures.
In this passage we have a citation linking to another paper hosted on the CDC's website. This one is considerably more concise in comparison, but that could merely be due to the relative simplicity of the subject covered.

Abstract​


The etiologic agent of an outbreak of pneumonia in Wuhan, China, was identified as severe acute respiratory syndrome coronavirus 2 in January 2020. A patient in the United States was given a diagnosis of infection with this virus by the state of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens from this patient and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into 2 virus repositories, making it broadly available to the public health and research communities. We hope that open access to this reagent will expedite development of medical countermeasures.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as the source of a pneumonia outbreak in Wuhan, China, in late 2019 (1,2). The virus was found to be a member of the β coronavirus family, in the same species as SARS-CoV and SARS-related bat CoVs (3,4). Patterns of spread indicate that SARS-CoV-2 can be transmitted person-to-person, and may be more transmissible than SARS-CoV (57). The spike protein of coronaviruses mediates virus binding and cell entry. Initial characterization of SARS-CoV-2 spike indicates that it binds the same receptor as SARS-CoV angiotensin-converting enzyme, which is expressed in both upper and lower human respiratory tracts (8).
The unprecedented rapidity of spread of this outbreak represents a critical need for reference reagents. The public health community requires viral lysates to serve as diagnostic references, and the research community needs virus isolates to test antiviral compounds, develop new vaccines, and perform basic research. In this article, we describe isolation of SARS-CoV-2 from a patient who had coronavirus disease (COVID-19) in the United States and described its genomic sequence and replication characteristics. We have made the virus isolate available to the public health community by depositing it into 2 virus reagent repositories.

Methods​

Specimen Collection​

Virus isolation from patient samples was deemed not to be human subjects research by the National Center for Immunizations and Respiratory Diseases, Centers for Disease Control and Prevention (CDC) (research determination no. 0900f3eb81ab4b6e). Clinical specimens from a case-patient who had acquired COVID-19 during travel to China and who was identified in Washington, USA, were collected as described (1). Nasopharyngeal (NP) and oropharyngeal (OP) swab specimens were collected on day 3 postsymptom onset, placed in 2–3 mL of viral transport medium, used for molecular diagnosis, and frozen. Confirmed PCR-positive specimens were aliquoted and refrozen until virus isolation was initiated.

Cell Culture, Limiting Dilution, and Virus Isolation​

We used Vero CCL-81 cells for isolation and initial passage. We cultured Vero E6, Vero CCL-81, HUH 7.0, 293T, A549, and EFKB3 cells in Dulbecco minimal essential medium (DMEM) supplemented with heat-inactivated fetal bovine serum (5% or 10%) and antibiotics/antimycotics (GIBCO, https://www.thermofisher.comExternal Link). We used both NP and OP swab specimens for virus isolation. For isolation, limiting dilution, and passage 1 of the virus, we pipetted 50 μL of serum-free DMEM into columns 2–12 of a 96-well tissue culture plate, then pipetted 100 μL of clinical specimens into column 1 and serially diluted 2-fold across the plate. We then trypsinized and resuspended Vero cells in DMEM containing 10% fetal bovine serum, 2× penicillin/streptomycin, 2× antibiotics/antimycotics, and 2× amphotericin B at a concentration of 2.5 × 105 cells/mL. We added 100 μL of cell suspension directly to the clinical specimen dilutions and mixed gently by pipetting. We then grew the inoculated cultures in a humidified 37°C incubator in an atmosphere of 5% CO2 and observed for cytopathic effects (CPEs) daily. We used standard plaque assays for SARS-CoV-2, which were based on SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) protocols (9,10).
When CPEs were observed, we scraped cell monolayers with the back of a pipette tip. We used 50 μL of viral lysate for total nucleic acid extraction for confirmatory testing and sequencing. We also used 50 μL of virus lysate to inoculate a well of a 90% confluent 24-well plate.

Inclusivity/Exclusivity Testing​

From the wells in which CPEs were observed, we performed confirmatory testing by using real-time reverse transcription PCR (CDC) and full-genome sequencing (1). The CDC molecular diagnostic assay targets 3 portions of the nucleocapsid gene, and results for all 3 portions must be positive for a sample to be considered positive (https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-detection-instructions.html and https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html). To confirm that no other respiratory viruses were present, we performed Fast Track Respiratory Pathogens 33 Testing (FTD Diagnostics, http://www.fast-trackdiagnostics.comExternal Link).

Whole-Genome Sequencing​

We designed 37 pairs of nested PCRs spanning the genome on the basis of the coronavirus reference sequence (GenBank accession no. NC045512). We extracted nucleic acid from isolates and amplified by using the 37 individual nested PCRs. We used positive PCR amplicons individually for subsequent Sanger sequencing and also pooled them for library preparation by using a ligation sequencing kit (Oxford Nanopore Technologies, https://nanoporetech.comExternal Link), subsequently for Oxford Nanopore MinION sequencing. We generated consensus nanopore sequences by using Minimap version 2.17 (https://github.comExternal Link) and Samtools version 1.9 (http://www.htslib.orgExternal Link). We generated consensus sequences by Sanger sequencing from both directions by using Sequencher version 5.4.6 (https://www.genecodes.comExternal Link), and further confirmed them by using consensus sequences generated from nanopore sequencing.
To sequence passage 4 stock, we prepared libraries for sequencing by using the Next Ultra II RNA Prep Kit (New England Biolabs, https://www.neb.comExternal Link) according to the manufacturer’s protocol. In brief, we fragmented ≈70–100 ng of RNA for 15 min, followed by cDNA synthesis, end repair, and adaptor ligation. After 6 rounds of PCR, we analyzed libraries by using an Agilent Bioanalyzer (https://www.agilent.comExternal Link) and quantified them by using a quantitative PCR. We pooled samples and sequenced samples by using a paired-end 75-base protocol on an Illumina (Illumina, Inc., https://www.illumina.comExternal Link) MiniSeq instrument and using the High-Output Kit and then processed reads by using Trimmomatic version 0.36 (11) to remove low-quality base calls and any adaptor sequences. We used the de novo assembly program ABySS (12) to assemble the reads into contigs by using several different sets of reads and kmer values ranging from 20 to 40. We compared contigs >400 bases against the National Center for Biotechnology Information (Bethesda, MD, USA) nucleotide collection using BLAST (https://blast.ncbi.nlm.nih.govExternal Link). A nearly full-length viral contig obtained in each sample had 100% identity to the 2019-nCoV/USA-WA1/2020 strain (GenBank accession no. MN985325.1). All the remaining contigs mapped to either host cell rRNA or mitochondria. We mapped the trimmed reads to the reference sequence by using BWA version 0.7.17 (13) and visualized these reads by using the Integrated Genomics Viewer (14) to confirm the identity with the USA-WA1/2020 strain.

Electron Microscopy​

We scraped infected Vero cells from the flask, pelleted by low-speed centrifugation, rinsed with 0.1 mol/L phosphate buffer, pelleted again, and fixed for 2 h in 2.5% buffered glutaraldehyde. We then postfixed specimens with 1% osmium tetroxide, en bloc stained with 4% uranyl acetate, dehydrated, and embedded in epoxy resin. We cut ultrathin sections, stained them with 4% uranyl acetate and lead citrate, and examined them by using a Thermo Fisher/FEI Tecnai Spirit electron microscope (https://www.fei.comExternal Link).

Protein Analysis and Western Blotting​

We harvested cell lysates by using Laemmli sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer (Bio-Rad, https://www.bio-rad.comExternal Link) containing 2% SDS and 5% β-mercaptoethanol. We removed the cell lysates from a Biosafety Level 3 Laboratory, boiled them, and load them onto a polyacrylamide gel. We subjected the lysates to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by transfer to a polyvinylidene difluoride polyvinylidene fluoride membrane. We then blocked the membrane in 5% nonfat dry milk dissolved in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 1 h, followed by a short wash with TBS-T. We incubated the membrane overnight with primary antibody, either rabbit polyclonal serum against the SARS-CoV spike protein (#40150-T52; Sino Biological, https://www.sinobiological.comExternal Link), β-actin antibody (#4970; Cell Signaling Technology, https://www.cellsignal.comExternal Link), or a custom rabbit polyclonal serum against SARS-CoV nucleocapsid. We then washed the membrane with 3 times with TBS-T and applied horseradish peroxidase-conjugated secondary antibody for 1 h. Subsequently, we washed the membrane 3 times with TBS-T, incubated with Clarity Western ECL Substrate (#1705060S; Bio-Rad), and imaged with a multipurpose imaging system.

Generation of SARS-CoV Nucleocapsid Antibodies​

We used the plasmid pBM302 (15) to express SARS-CoV nucleocapsid protein, with a C-terminal His6 tag, to high levels within the inclusion bodies of Escherichia coli and the recombinant protein was purified from the inclusion bodies by using nickel-affinity column chromatography under denaturing conditions. We used stepwise dialysis against Tris/phosphate buffer to refold the recombinant SARS-CoV nucleocapsid protein with decreasing concentrations of urea to renature the protein. We then immunized rabbits with the renatured, full-length, SARS-CoV nucleocapsid protein to generate an affinity-purified rabbit anti–SARS-CoV nucleocapsid protein polyclonal antibody.

Results​

Thumbnail of Cytopathic effect caused by severe acute respiratory syndrome coronavirus 2 from patient with 2019 novel coronavirus disease, United States, 2020. A–C) Phase-contrast microscopy of Vero cell monolayers at 3 days postinoculation: A) Mock, B) nasopharyngeal specimen, C) oropharyngeal specimen. Original magnifications ×10). D) Electron microscopy of virus isolate showing extracellular spherical particles with cross-sections through the nucleocapsids (black dots). Arrow indicates a coro

Figure 1. Cytopathic effect caused by severe acute respiratory syndrome coronavirus 2 from patient with coronavirus disease, United States, 2020. A–C) Phase-contrast microscopy of Vero cell monolayers at 3 days postinoculation: A) Mock,...
A patient was identified with confirmed COVID-19 in Washington State on January 22, 2020. CPE was not observed in mock infected cells (Figure 1, panel A). Cycle threshold (Ct) values were 18–20 for NP specimens and 21–22 for OP specimens (1). The positive clinical specimens were aliquoted and refrozen inoculated into cell culture on January 22, 2020. We observed CPE 2 days postinoculation and harvested viral lysate on day 3 postinoculation (Figure 1, panels B, C). We used 50 μL of passage 1 viral lysates for nucleic acid extraction to confirm the presence of SARS-CoV-2 by using the CDC molecular diagnostic assay (1). The Ct values of 3 nucleic acid extractions were 16.0–17.1 for nucleocapsid portion 1, 15.9–17.1 for nucleocapsid portion 2, and 16.2–17.3 for nucleocapsid portion 3, which confirmed isolation of SARS-CoV-2 (Ct <40 is considered a positive result). We also tested extracts for 33 additional different respiratory pathogens by using the Fast Track 33 Assay. No other pathogens were detected. Identity was additionally supported by thin-section electron microscopy (Figure 1, panel D). We observed a morphology and morphogenesis characteristic of coronaviruses.
We used isolates from the first passage of an OP and an NP specimen for whole-genome sequencing. The genomes from the NP specimen (GenBank accession MT020880) and OP specimen (GenBank accession no. MT020881) showed 100% identity with each other. The isolates also showed 100% identity with the corresponding clinical specimen (GenBank accession no. MN985325).
After the second passage, we did not culture OP and NP specimens separately. We passaged virus isolate 2 more times in Vero CCL-81 cells and titrated by determining the 50% tissue culture infectious dose (TCID50). Titers were 8.65 × 106 TCID50/mL for the third passage and 7.65 × 106 TCID50/mL for the fourth passage.
We passaged this virus in the absence of trypsin. The spike protein sequence of SARS-CoV-2 has an RRAR insertion at the S1-S2 interface that might be cleaved by furin (16). Highly pathogenic avian influenza viruses have highly basic furin cleavage sites at the hemagglutinin protein HA1-HA2 interface that permit intracellular maturation of virions and more efficient viral replication (17). The RRAR insertion in SARS-CoV-2 might serve a similar function.
Thumbnail of Viral propagation and quantitation of severe acute respiratory syndrome coronavirus 2 from patient with 2019 novel coronavirus disease, United States, 2020. A) Two virus passage 4 stocks (black and gray circles) were quantified by using plaque assay at day 2 (solid circles) and day 3 (open circles) postinfection of Vero E6 and Vero CCL81 cells. B) Plaque morphology for virus on Vero E6 and Vero CCL81 at day 2 and day 3 postinoculation. C) Cell monolayers 2 days postinfection of Vero

Figure 2. Viral propagation and quantitation of severe acute respiratory syndrome coronavirus 2 from patient with coronavirus disease, United States, 2020. A) Two virus passage 4 stocks (black and gray circles) were quantified...
We subsequently generated a fourth passage stock of SARS-CoV-2 on VeroE6 cells, another fetal rhesus monkey kidney cell line. We sequenced viral RNA from SARS-CoV-2 passage 4 stock and confirmed it to have no nucleotide mutations compared with the original reference sequence (GenBank accession no. MN985325). SARS-CoV has been found to grow well on VeroE6 cells and MERS-CoV on Vero CCL81 cells (18,19). To establish a plaque assay and determine the preferred Vero cell type for quantification, we titered our passage 4 stock on VeroE6 and VeroCCL81 cells. After infection with a dilution series, SARS-CoV-2 replicated in both Vero cell types; however, the viral titers were slightly higher in VeroE6 cells than in Vero CCL81 cells (Figure 2, panel A). In addition, plaques were more distinct and visible on Vero E6 cells (Figure 2, panel B). As early as 2 days postinoculation, VeroE6 cells produced distinct plaques visible by staining with neutral red. In contrast, Vero CCL81 cells produced less clear plaques and was most easily quantitated by staining with neutral red 3 days postinoculation. On the individual plaque monolayers, SARS-CoV-2 infection of Vero E6 cells produced CPE with areas of cell clearance (Figure 2, panel C). In contrast, Vero CCL81 cells had areas of dead cells that had fused to form plaques, but the cells did not clear. Together, these results suggest that VeroE6 cells might be the best choice for amplification and quantification, but both Vero cell types support amplification and replication of SARS-CoV-2.
Thumbnail of Cell lines from patient with coronavirus disease, United States, 2020, susceptible to SARS coronavirus 2 (SARS-CoV-2). Cell lines were infected with a high multiplicity of infection (>5), washed after adsorption, and subsequently harvested 24 h postinfection for viral titer and protein lysates. A) Viral titer for SARS-CoV-2 quantitated by plaque assay on Vero E6 cells 2 days postinoculation. Infected cell protein lysates were probed by using Western blotting with B) rabbit polycl

Figure 3. Cell lines from patient with coronavirus disease, United States, 2020, susceptible to SARS coronavirus 2 (SARS-CoV-2). Cell lines were infected with a high multiplicity of infection (>5), washed after adsorption, and...
Because research has been initiated to study and respond to SARS-CoV-2, information about cell lines and types susceptible to infection is needed. Therefore, we examined the capacity of SARS-CoV-2 to infect and replicate in several common primate and human cell lines, including human adenocarcinoma cells (A549), human liver cells (HUH7.0), and human embryonic kidney cells (HEK-293T), in addition to Vero E6 and Vero CCL81 cells. We also examined an available big brown bat kidney cell line (EFK3B) for SARS-CoV-2 replication capacity. Each cell line was inoculated at high multiplicity of infection and examined 24 h postinfection (Figure 3, panel A). No CPE was observed in any of the cell lines except in Vero cells, which grew to >107 PFU at 24 h postinfection. In contrast, HUH7.0 and 293T cells showed only modest viral replication, and A549 cells were incompatible with SARS-CoV-2 infection. These results are consistent with previous susceptibility findings for SARS-CoV and suggest other common culture systems, including MDCK, HeLa, HEP-2, MRC-5 cells, and embryonated eggs, are unlikely to support SARS-CoV-2 replication (2022). In addition, SARS-CoV-2 did not replicate in bat EFK3B cells, which are susceptible to MERS-CoV. Together, the results indicate that SARS-CoV-2 maintains a similar profile to SARS-CoV in terms of susceptible cell lines.
Having established robust infection with SARS-CoV-2 in several cell types, we next evaluated the cross-reactivity of SARS-CoV antibodies against the SARS-CoV-2. Cell lysates from infected cell lines were probed for protein analysis; we found that polyclonal serum against the SARS-CoV spike protein and nucleocapsid proteins recognize SARS-CoV-2 (Figure 3, panels B, C). The nucleocapsid protein, which is highly conserved across the group 2B family, retains >90% amino acid identity between SARS-CoV and SARS-CoV-2. Consistent with the replication results (Figure 3, panel A), SARS-CoV-2 showed robust nucleocapsid protein in both Vero cell types, less protein in HUH7.0 and 293T cells, and minimal protein in A549 and EFK3B cells (Figure 3, panel B). The SARS-CoV spike protein antibody also recognized SARS-CoV-2 spike protein, indicating cross-reactivity (Figure 3, panel C). Consistent with SARS CoV, several cleaved and uncleaved forms of the SARS-CoV-2 spike protein were observed. The cleavage pattern of the SARS spike positive control from Calu3 cells, a respiratory cell line, varies slightly and could indicate differences between proteolytic cleavage of the spike proteins between the 2 viruses because of a predicted insertion of a furin cleavage site in SARS-CoV-2 (16). However, differences in cell type and conditions complicate this interpretation and indicate the need for further study in equivalent systems. Overall, the protein expression data from SARS-CoV nucleocapsid and spike protein antibodies recapitulate replication findings and indicate that SARS-CoV reagents can be used to characterize SARS-CoV-2 infection.
Thumbnail of Multistep growth curve for severe acute respiratory syndrome coronavirus 2 from patient with 2019 novel coronavirus disease, United States, 2020. Vero CCL81 (black) and HUH7.0 cells (green) were infected at a multiplicity of infection of 0.1, and cells (solid line) and supernatants (dashed line) were harvested and assayed for viral replication by using TCID50. Circles, Vero CCL81 cells; squares, Vero CCL81 supernatants; triangles, HUH7.0 cells; inverted triangles, HUH7.0 supernatant

Figure 4. Multistep growth curve for severe acute respiratory syndrome coronavirus 2 from patient with coronavirus disease, United States, 2020. Vero CCL81 (black) and HUH7.0 cells (green) were infected at a multiplicity of...
Finally, we evaluated the replication kinetics of SARS-CoV-2 in a multistep growth curve. In brief, we infected Vero CCL-81 and HUH7.0 cells with SARS-CoV-2 at a low multiplicity of infection (0.1) and evaluated viral replication every 6 h for 72 h postinoculation, with separate harvests in the cell-associated and supernatant compartments (Figure 4). Similar to SARS-CoV, SARS-CoV-2 replicated rapidly in Vero cells after an initial eclipse phase, achieving 105 TCID50/mL by 24 h postinfection and peaking at >106 TCID50/mL. We observed similar titers in cell-associated and supernatant compartments, which indicated efficient egress. Despite peak viral titers by 48 h postinoculation, major CPE was not observed until 60 h postinoculation and peaked at 72 h postinoculation, indicating that infected monolayers should be harvested before peak CPE is observed. Replication in HUH7.0 cells also increased quickly after an initial eclipse phase but plateaued by 24 h postinoculation in the intracellular compartment at 2 × 103 TCID50/mL and decreased after 66 h postinoculation. Virus was not detected in the supernatant of infected HUH7 cells until 36 h postinoculation and exhibited lower titers at all timepoints (Figure 4). Major CPE was never observed in HUH7.0 cells. These results are consistent with previous reports for SARS-CoV and MERS-CoV, which suggested similar replication dynamics between the zoonotic CoV strains (23,24).

Discussion​

We have deposited information on the SARS-CoV-2 USA-WA1/2020 viral strain described here into the Biodefense and Emerging Infections Research Resources Repository (https://www.beiresources.orgExternal Link) reagent resources (American Type Culture Collection, https://www.atcc.orgExternal Link) and the World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch (https://www.utmb.edu/wrcevaExternal Link), to serve as the SARS-CoV-2 reference strain for the United States. The SARS-CoV-2 fourth passage virus has been sequenced and maintains a nucleotide sequence identical to that of the original clinical strain from the United States. These deposits make this virus strain available to the domestic and international public health, academic, and pharmaceutical sectors for basic research, diagnostic development, antiviral testing, and vaccine development. We hope broad access will expedite countermeasure development and testing and enable a better understanding of the transmissibility and pathogenesis of this novel emerging virus.
It's pretty dense and is apparently evidence against the methods used by virologists, so I may have to cover it in another autism sidequest. But for now, that's the end of this section and the end of my post. Here's the next segment in plain text:
He who controls the language controls the masses. — Saul Alinsky

A further embarrassment for virology is that alleged viral particles that have been successfully purified have not been shown to be replication-competent or disease-causing by themselves. In other words, what have been physically isolated can only be said to be extracellular vesicles (EVs). In May 2020, a publication appeared in the journal Viruses that claimed, “nowadays, it is an almost impossible mission to separate EVs and viruses by means of canonical vesicle isolation methods, such as differenEal ultracentrifugation, because they are frequently co-pelleted due to their similar dimension.” ‘Nowadays’ means in contrast to the past and it is unclear how such an observed technical change may be reconciled with biological laws. It appears more likely that the virologists are distancing themselves from their own techniques in order to avoid refutation of their own postulates. They may have to accept that the reason differential ultracentrifugation is not able to separate viruses from other vesicles is because their assertion that viruses are present in the sample is ill-founded.

The virologists are clearly distracting from the foundational issue of isolaton as they have been unable to deliver on this front. Instead of addressing the problem honestly and scientifically, they have obfuscated the language. In 2017, The Perth Group pointed out in their magnum opus, “HIV - a virus like no other” that, “in virology, while purification retains its everyday meaning, ‘isolation’ is an expediential term virologists assign to data they claim are proof a particular virus exists.” In other words, it is convenient and practical but with regard to the claims that are made and the subsequent actions that are carried out against humanity, it should be viewed as improper and immoral. In the same essay, The Perth Group documented the following examples of virologists adapting the scientific language, as suited, for their own purposes:

HIV expert Jay Levy defines virus isolation as a "sample of a virus from a defined source", White and Fenner as the ability to "identify a totally unforeseen virus, or even discover an entirely new agent". Montagnier and Weiss as "propagating them [viruses] in cells in culture". The 2013 sixth edition of Fields Virology defines isolation as "Viruses can be isolated from an infected host by harvesting excreted or secreted material, blood, or tissue and testing for induction of the original symptoms in the identical host, or induction of some abnormal pathology in a substitute host, or in a cell culture...Once the presence of a virus has been established, it is oben desirable to prepare a genetically pure clone". It goes without saying that if virus isola6on is to "take a sample of a virus from a defined source", or "propagating them in cells in culture", one first must have proof the virus exists in "a defined source" or "in cells in culture". Neither is virus isolation "induc6on of some abnormal pathology" or "once the presence of a virus
has been established".

It is a travesty that this state of affairs exists and the grossly misleading practice renders virology’s many claims of isolation as unsubstantiated. But do the virologists themselves offer any explanation for their relentless abuse of the English language? In 2021, veteran virologist Professor Vincent Racaniello explained, even with regard to the definition of fundamental terms such as ‘isolate’ that, “what happens is you’re trained in someone’s laboratory and you hear them say things and you associate a meaning with them and that’s what you do, and they may or may not be right.” In the same presentation, Racaniello himself didn’t appear to notice a problem with his own definition of what are supposed to be scientific terms when he went on to say, “an isolate is a virus that we have isolated from an infected host and we have propagated that in culture.” Ironically, in a 2015 article, regarding appropriate scientific terminology and the word ‘transfection’, Racaniello stated, “if you view the English language as a dynamic means of communication that continually evolves and provides words with new meanings, then this incorrect use of transfection probably does not bother you. But scientists must be precise in their use of language, otherwise their ability to communicate will be impaired.” An analysis of Racaniello's presentation on viral isolation and the misuse of language in science has been dealt with previously by Dr Samantha Bailey in, “The Truth About Virus Isolation.” It is illustrative of the problem where multiple generations of virologists appear trapped in a world of semantic circular reasoning, albeit with differing degrees of insight.

Virology invented the hypothesis of viruses so whatever method it employs in an attempt to prove their existence, it must satisfy that definition. At the heart of the matter is a simple concept and we need to see evidence that alleged disease-causing particles cause new particles that are clones of the former. Claiming that detected proteins and nucleic acids are of a specific viral origin is not possible unless the alleged viral particles have been truly isolated by purification and shown to have these key biological characteristics. As outlined by The Perth Group in, “HIV - a virus like no other,” purification is necessary to prove the existence of viruses for several reasons, including the following:

1. Viruses replicate only in living cells. Since cells and viruses are composed of the same biochemical constituents, separation of particles from cellular material is essential for defining which nucleic acid and proteins belong to the virus particles.

2. To prove the particles are infectious. In other words, it is particles, not other factors, that are responsible for the production of new particles. This requires purification of both sets of particles.

3. To demonstrate their biological and pathological effects.

4. To obtain antigens (proteins) and nucleic acids for use in antibody and genomic tests respectively.

Although less common, virologists will sometimes obfuscate the meaning of ‘purification’ as well. On 23 May 2022, Belgian Professor of Virology Marc Van Ranst claimed that with regard to SARS-30 CoV-2, “in another article (hgps://europepmc.org/article/pmc/pmc7122600) they have further purified the virus by ultracentrifugation in beta-cyclodextrin.” Van Ranst was referring to a 2008 paper that described, “large-Scale preparation of UV-Inactivated SARS coronavirus virions,” which related to the purported SARS-CoV-1 virus. However, this paper simply outlines a protocol claiming to purify virions and there is no part of the paper that demonstrated the existence of any replication-competent particle — all that was shown were some low quality images purporting to show “infected” Vero E6 cells. (See next section regarding ‘cytopathic effects’.) With regard to the “check of purified virions” following centrifugation, no images were provided but the claim was made that, “the concentration of purified virions is determined by BCA [bicinchoninic acid] assay with BSA [bovine serum albumin] as a standard.” This was an unfounded conclusion as the BCA assay simply measures the total concentration of protein in a solution — the technique is unable to provide evidence that there are any “virions” present in a sample.

Figure 1 below is an image purporting to show purified “bat SARS-like coronavirus” virions and was published in Nature in 2013 — the caption explains why such a declaration is ludicrous. (The convenient variation in particle size is apparently because, “[coronaviruses] usually have a diameter, excluding projections, of between 80 and 120 nm, although in extreme cases the diameter can vary between 60 and 220 nm.” ) Likewise, the claim in Van Ranst’s cited paper that, “it is beger to confirm the amount of virion by 10% SDS-PAGE,” is just as erroneous as this is simply a gel electrophoresis process to separate out proteins by their molecular mass — it cannot provide evidence that the proteins belong to a virus. Van Ranst also stated, “we can already detect the viral RNA in clinical samples. We can complete the viral genome decipher. We can grow the virus in cell culture and inoculate it into animal models and induce disease.” It is unknown whether Van Ranst appreciated that the uncontrolled methodologies being employed in all such experiments do not provide the required evidence for any “virus.” So, when Van Ranst made the claim that, “no scientist doubts the existence of SARS-CoV-2,” it makes one wonder whether the virologists will now have to change the definition of ‘scientist’ to maintain the delusive practices?

Van Ranst was not the only virologist making claims about purifying viruses though. In response to an email enquiry, Dr Marica Grossegesse from the Robert Koch Institute responded that, “we purified SARS particles by density gradient. However, just from the cell culture derived virus, as you wrote. The challenge with purifying SARS from patient samples is that you won’t get a visible band.” Apart from the imprecise terminology in substituting the name of a syndrome (‘SARS’ is severe acute respiratory syndrome) for a postulated virus, no further evidence was supplied as to how these claims were established. Presumably, Grossegesse is also using the definitions of “purification” and “virus” as depicted in Figure 1? In any case, when pressed for further details about how the experiments were controlled she responded, “we are not allowed to share any protocols with a private person. I can only refer to our publications, where infection experiments are described in detail.” It appears that ‘detail’ has taken on a different meaning as well, as the publications failed to disclose the straightforward answers concerning controls being sought.

The area of isolation is one of the domains where virology is completely unhinged and as this essay will outline, SARS-CoV-2 remains nothing more than a hypothetical computer construct, assembled from genetic fragments of unproven provenance. There has never been a physically isolated (i.e. purified) particle shown to be responsible for the production of identical particles or a particle shown to be the cause of pathological effects in any human or in an experimental animal model. Thus, the declaration by virologists such as Van Ranst, along with the WHO and its adherents, that an infectious particle termed ‘SARS-CoV-2’ is causing a disease pandemic is shown to be patent scientific and intellectual fraud.
It's a doozy, so I may have to chop that one up into parts as well.
 
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the first screenshot
The first screenshot is replicated in full in the quote beneath it, and is taken from the same page you provided as a citation. Your own citation therefore disproves your claim, as it goes to great lengths to explain why your assertions about the meaning of "theory", in the context of scientific discussion, are wrong.

I repeat: You are an ignorant faggot. Illiterate, too.
 
The first screenshot is replicated in full in the quote beneath it, and is taken from the same page you provided as a citation. Your own citation therefore disproves your claim, as it goes to great lengths to explain why your assertions about the meaning of "theory", in the context of scientific discussion, are wrong.

I repeat: You are an ignorant faggot. Illiterate, too.

Nope. You, in fact, are the illiterate ignorant faggot here and I already explained why. Your "proof" only supports what I said.
 
As for "Essue culture experiments" I'll defer to people who know what the hell that means.
It's a typo of tissue culture. Essue isn't a term. Tissue culture is simply a procedure where you artificially grow a tissue or cell type outside of the organism in sterile flasks with media specially tailored for whatever your goal for the cells are. (usually just to grow them efficiently to have more of a particular cell type)
 
It's a typo of tissue culture. Essue isn't a term. Tissue culture is simply a procedure where you artificially grow a tissue or cell type outside of the organism in sterile flasks with media specially tailored for whatever your goal for the cells are. (usually just to grow them efficiently to have more of a particular cell type)
Oh okay. Copying from the PDF doesn't seem to render letter combinations like "ti" or "tt", so I've had to painstakingly go through and add them in myself.

I'm interested in feedback if you're willing to give any. Obviously you're fond of my posts seeing as you keep giving me reddit gold winner stickers whenever I make them and I'd continue making them even if I was getting trashcans and puzzle pieces.
 
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I'm interested in feedback if you're willing to give any. Obviously you're fond of my posts seeing as you keep giving me reddit gold winner stickers whenever I make them and I'd continue making them even if I was getting trashcans and puzzle pieces.
I'm enjoying the breakdowns focusing more on persuasiveness and appeals to emotion of the overall arguments instead of the more technical aspects, yeah. If you're worried about missing the context over not understanding certain scientific terms then you can keep a 2nd window open to just immediately search them as you read along. It helped me out a lot when I was starting to read papers myself a long time ago. (and the internet is a hell of a lot faster than thumbing through a paperback dictionary!)

It always makes me happy to see people outside of the field trying to read up and understand it. Especially these days since general trust in research is at an all-time low thanks to the political fuckery happening with the Covid-19 vaccine's development. The research fields need all of the scrutiny they can get now since the actually good papers will be able to handle it, and it will make everyone involved more sensitive and serious about what they publish.
 
it will make everyone involved more sensitive and serious about what they publish

Implying that wasn't the case before, yet so many insist otherwise. The Gell-Mann Amnesia Effect is alive and well.
 
It's a known fact that simply giving your attention to something can change it, you're right. But we don't even have to get into quantum physics, the very physical mechanical procedures that cells are subjected to in electron microscopy itself render the whole process as little more than a cultish ritual. It has the same level of scientific legitimacy as a witch pouring a bunch of rotting liquids into a cauldron and calling it a medicinal potion.
View attachment 5598146
Nice word salad, I'd invoke Poe's law, but you're known schizophrenic retard with delusions grandeur.


Lookie here viruses seen under electromicroscope

Concrete and undeniable proof that virii excist
 
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  • Informative
Reactions: HIVidaBoheme
ell oh ell.png
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Do you have a post that would perfectly fit this situation?
Congratulations! You've reached a point where people are just marking your posts out of sheer spite without actually reading anything you say.
you negrated me because you can't actually argue for or against this matter. Your mind is made up and so, in lieu of any critical thinking or the ability to read and understand, you just react.
Funny that you would have a post like that. Weird.
A post that would perfectly describe your own behavior, in this thread. Weird!
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Gay as it is to care about stickers, to go through all of my posts in this thread and give them stylish headwear out of sheer spite is like having a train run on you by an entire pride parade. We're nearing Bryan Dunn levels on the MATI scale.

Abstract​


The etiologic agent of an outbreak of pneumonia in Wuhan, China, was identified as severe acute respiratory syndrome coronavirus 2 in January 2020. A patient in the United States was given a diagnosis of infection with this virus by the state of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens from this patient and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into 2 virus repositories, making it broadly available to the public health and research communities. We hope that open access to this reagent will expedite development of medical countermeasures.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as the source of a pneumonia outbreak in Wuhan, China, in late 2019 (1,2). The virus was found to be a member of the β coronavirus family, in the same species as SARS-CoV and SARS-related bat CoVs (3,4). Patterns of spread indicate that SARS-CoV-2 can be transmitted person-to-person, and may be more transmissible than SARS-CoV (57). The spike protein of coronaviruses mediates virus binding and cell entry. Initial characterization of SARS-CoV-2 spike indicates that it binds the same receptor as SARS-CoV angiotensin-converting enzyme, which is expressed in both upper and lower human respiratory tracts (8).
The unprecedented rapidity of spread of this outbreak represents a critical need for reference reagents. The public health community requires viral lysates to serve as diagnostic references, and the research community needs virus isolates to test antiviral compounds, develop new vaccines, and perform basic research. In this article, we describe isolation of SARS-CoV-2 from a patient who had coronavirus disease (COVID-19) in the United States and described its genomic sequence and replication characteristics. We have made the virus isolate available to the public health community by depositing it into 2 virus reagent repositories.

Methods​

Specimen Collection​

Virus isolation from patient samples was deemed not to be human subjects research by the National Center for Immunizations and Respiratory Diseases, Centers for Disease Control and Prevention (CDC) (research determination no. 0900f3eb81ab4b6e). Clinical specimens from a case-patient who had acquired COVID-19 during travel to China and who was identified in Washington, USA, were collected as described (1). Nasopharyngeal (NP) and oropharyngeal (OP) swab specimens were collected on day 3 postsymptom onset, placed in 2–3 mL of viral transport medium, used for molecular diagnosis, and frozen. Confirmed PCR-positive specimens were aliquoted and refrozen until virus isolation was initiated.

Cell Culture, Limiting Dilution, and Virus Isolation​

We used Vero CCL-81 cells for isolation and initial passage. We cultured Vero E6, Vero CCL-81, HUH 7.0, 293T, A549, and EFKB3 cells in Dulbecco minimal essential medium (DMEM) supplemented with heat-inactivated fetal bovine serum (5% or 10%) and antibiotics/antimycotics (GIBCO, https://www.thermofisher.comExternal Link). We used both NP and OP swab specimens for virus isolation. For isolation, limiting dilution, and passage 1 of the virus, we pipetted 50 μL of serum-free DMEM into columns 2–12 of a 96-well tissue culture plate, then pipetted 100 μL of clinical specimens into column 1 and serially diluted 2-fold across the plate. We then trypsinized and resuspended Vero cells in DMEM containing 10% fetal bovine serum, 2× penicillin/streptomycin, 2× antibiotics/antimycotics, and 2× amphotericin B at a concentration of 2.5 × 105 cells/mL. We added 100 μL of cell suspension directly to the clinical specimen dilutions and mixed gently by pipetting. We then grew the inoculated cultures in a humidified 37°C incubator in an atmosphere of 5% CO2 and observed for cytopathic effects (CPEs) daily. We used standard plaque assays for SARS-CoV-2, which were based on SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) protocols (9,10).
When CPEs were observed, we scraped cell monolayers with the back of a pipette tip. We used 50 μL of viral lysate for total nucleic acid extraction for confirmatory testing and sequencing. We also used 50 μL of virus lysate to inoculate a well of a 90% confluent 24-well plate.

Inclusivity/Exclusivity Testing​

From the wells in which CPEs were observed, we performed confirmatory testing by using real-time reverse transcription PCR (CDC) and full-genome sequencing (1). The CDC molecular diagnostic assay targets 3 portions of the nucleocapsid gene, and results for all 3 portions must be positive for a sample to be considered positive (https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-detection-instructions.html and https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html). To confirm that no other respiratory viruses were present, we performed Fast Track Respiratory Pathogens 33 Testing (FTD Diagnostics, http://www.fast-trackdiagnostics.comExternal Link).

Whole-Genome Sequencing​

We designed 37 pairs of nested PCRs spanning the genome on the basis of the coronavirus reference sequence (GenBank accession no. NC045512). We extracted nucleic acid from isolates and amplified by using the 37 individual nested PCRs. We used positive PCR amplicons individually for subsequent Sanger sequencing and also pooled them for library preparation by using a ligation sequencing kit (Oxford Nanopore Technologies, https://nanoporetech.comExternal Link), subsequently for Oxford Nanopore MinION sequencing. We generated consensus nanopore sequences by using Minimap version 2.17 (https://github.comExternal Link) and Samtools version 1.9 (http://www.htslib.orgExternal Link). We generated consensus sequences by Sanger sequencing from both directions by using Sequencher version 5.4.6 (https://www.genecodes.comExternal Link), and further confirmed them by using consensus sequences generated from nanopore sequencing.
To sequence passage 4 stock, we prepared libraries for sequencing by using the Next Ultra II RNA Prep Kit (New England Biolabs, https://www.neb.comExternal Link) according to the manufacturer’s protocol. In brief, we fragmented ≈70–100 ng of RNA for 15 min, followed by cDNA synthesis, end repair, and adaptor ligation. After 6 rounds of PCR, we analyzed libraries by using an Agilent Bioanalyzer (https://www.agilent.comExternal Link) and quantified them by using a quantitative PCR. We pooled samples and sequenced samples by using a paired-end 75-base protocol on an Illumina (Illumina, Inc., https://www.illumina.comExternal Link) MiniSeq instrument and using the High-Output Kit and then processed reads by using Trimmomatic version 0.36 (11) to remove low-quality base calls and any adaptor sequences. We used the de novo assembly program ABySS (12) to assemble the reads into contigs by using several different sets of reads and kmer values ranging from 20 to 40. We compared contigs >400 bases against the National Center for Biotechnology Information (Bethesda, MD, USA) nucleotide collection using BLAST (https://blast.ncbi.nlm.nih.govExternal Link). A nearly full-length viral contig obtained in each sample had 100% identity to the 2019-nCoV/USA-WA1/2020 strain (GenBank accession no. MN985325.1). All the remaining contigs mapped to either host cell rRNA or mitochondria. We mapped the trimmed reads to the reference sequence by using BWA version 0.7.17 (13) and visualized these reads by using the Integrated Genomics Viewer (14) to confirm the identity with the USA-WA1/2020 strain.

Electron Microscopy​

We scraped infected Vero cells from the flask, pelleted by low-speed centrifugation, rinsed with 0.1 mol/L phosphate buffer, pelleted again, and fixed for 2 h in 2.5% buffered glutaraldehyde. We then postfixed specimens with 1% osmium tetroxide, en bloc stained with 4% uranyl acetate, dehydrated, and embedded in epoxy resin. We cut ultrathin sections, stained them with 4% uranyl acetate and lead citrate, and examined them by using a Thermo Fisher/FEI Tecnai Spirit electron microscope (https://www.fei.comExternal Link).

Protein Analysis and Western Blotting​

We harvested cell lysates by using Laemmli sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer (Bio-Rad, https://www.bio-rad.comExternal Link) containing 2% SDS and 5% β-mercaptoethanol. We removed the cell lysates from a Biosafety Level 3 Laboratory, boiled them, and load them onto a polyacrylamide gel. We subjected the lysates to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by transfer to a polyvinylidene difluoride polyvinylidene fluoride membrane. We then blocked the membrane in 5% nonfat dry milk dissolved in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 1 h, followed by a short wash with TBS-T. We incubated the membrane overnight with primary antibody, either rabbit polyclonal serum against the SARS-CoV spike protein (#40150-T52; Sino Biological, https://www.sinobiological.comExternal Link), β-actin antibody (#4970; Cell Signaling Technology, https://www.cellsignal.comExternal Link), or a custom rabbit polyclonal serum against SARS-CoV nucleocapsid. We then washed the membrane with 3 times with TBS-T and applied horseradish peroxidase-conjugated secondary antibody for 1 h. Subsequently, we washed the membrane 3 times with TBS-T, incubated with Clarity Western ECL Substrate (#1705060S; Bio-Rad), and imaged with a multipurpose imaging system.

Generation of SARS-CoV Nucleocapsid Antibodies​

We used the plasmid pBM302 (15) to express SARS-CoV nucleocapsid protein, with a C-terminal His6 tag, to high levels within the inclusion bodies of Escherichia coli and the recombinant protein was purified from the inclusion bodies by using nickel-affinity column chromatography under denaturing conditions. We used stepwise dialysis against Tris/phosphate buffer to refold the recombinant SARS-CoV nucleocapsid protein with decreasing concentrations of urea to renature the protein. We then immunized rabbits with the renatured, full-length, SARS-CoV nucleocapsid protein to generate an affinity-purified rabbit anti–SARS-CoV nucleocapsid protein polyclonal antibody.

Results​

Thumbnail of Cytopathic effect caused by severe acute respiratory syndrome coronavirus 2 from patient with 2019 novel coronavirus disease, United States, 2020. A–C) Phase-contrast microscopy of Vero cell monolayers at 3 days postinoculation: A) Mock, B) nasopharyngeal specimen, C) oropharyngeal specimen. Original magnifications ×10). D) Electron microscopy of virus isolate showing extracellular spherical particles with cross-sections through the nucleocapsids (black dots). Arrow indicates a coro

Figure 1. Cytopathic effect caused by severe acute respiratory syndrome coronavirus 2 from patient with coronavirus disease, United States, 2020. A–C) Phase-contrast microscopy of Vero cell monolayers at 3 days postinoculation: A) Mock,...
A patient was identified with confirmed COVID-19 in Washington State on January 22, 2020. CPE was not observed in mock infected cells (Figure 1, panel A). Cycle threshold (Ct) values were 18–20 for NP specimens and 21–22 for OP specimens (1). The positive clinical specimens were aliquoted and refrozen inoculated into cell culture on January 22, 2020. We observed CPE 2 days postinoculation and harvested viral lysate on day 3 postinoculation (Figure 1, panels B, C). We used 50 μL of passage 1 viral lysates for nucleic acid extraction to confirm the presence of SARS-CoV-2 by using the CDC molecular diagnostic assay (1). The Ct values of 3 nucleic acid extractions were 16.0–17.1 for nucleocapsid portion 1, 15.9–17.1 for nucleocapsid portion 2, and 16.2–17.3 for nucleocapsid portion 3, which confirmed isolation of SARS-CoV-2 (Ct <40 is considered a positive result). We also tested extracts for 33 additional different respiratory pathogens by using the Fast Track 33 Assay. No other pathogens were detected. Identity was additionally supported by thin-section electron microscopy (Figure 1, panel D). We observed a morphology and morphogenesis characteristic of coronaviruses.
We used isolates from the first passage of an OP and an NP specimen for whole-genome sequencing. The genomes from the NP specimen (GenBank accession MT020880) and OP specimen (GenBank accession no. MT020881) showed 100% identity with each other. The isolates also showed 100% identity with the corresponding clinical specimen (GenBank accession no. MN985325).
After the second passage, we did not culture OP and NP specimens separately. We passaged virus isolate 2 more times in Vero CCL-81 cells and titrated by determining the 50% tissue culture infectious dose (TCID50). Titers were 8.65 × 106 TCID50/mL for the third passage and 7.65 × 106 TCID50/mL for the fourth passage.
We passaged this virus in the absence of trypsin. The spike protein sequence of SARS-CoV-2 has an RRAR insertion at the S1-S2 interface that might be cleaved by furin (16). Highly pathogenic avian influenza viruses have highly basic furin cleavage sites at the hemagglutinin protein HA1-HA2 interface that permit intracellular maturation of virions and more efficient viral replication (17). The RRAR insertion in SARS-CoV-2 might serve a similar function.
Thumbnail of Viral propagation and quantitation of severe acute respiratory syndrome coronavirus 2 from patient with 2019 novel coronavirus disease, United States, 2020. A) Two virus passage 4 stocks (black and gray circles) were quantified by using plaque assay at day 2 (solid circles) and day 3 (open circles) postinfection of Vero E6 and Vero CCL81 cells. B) Plaque morphology for virus on Vero E6 and Vero CCL81 at day 2 and day 3 postinoculation. C) Cell monolayers 2 days postinfection of Vero

Figure 2. Viral propagation and quantitation of severe acute respiratory syndrome coronavirus 2 from patient with coronavirus disease, United States, 2020. A) Two virus passage 4 stocks (black and gray circles) were quantified...
We subsequently generated a fourth passage stock of SARS-CoV-2 on VeroE6 cells, another fetal rhesus monkey kidney cell line. We sequenced viral RNA from SARS-CoV-2 passage 4 stock and confirmed it to have no nucleotide mutations compared with the original reference sequence (GenBank accession no. MN985325). SARS-CoV has been found to grow well on VeroE6 cells and MERS-CoV on Vero CCL81 cells (18,19). To establish a plaque assay and determine the preferred Vero cell type for quantification, we titered our passage 4 stock on VeroE6 and VeroCCL81 cells. After infection with a dilution series, SARS-CoV-2 replicated in both Vero cell types; however, the viral titers were slightly higher in VeroE6 cells than in Vero CCL81 cells (Figure 2, panel A). In addition, plaques were more distinct and visible on Vero E6 cells (Figure 2, panel B). As early as 2 days postinoculation, VeroE6 cells produced distinct plaques visible by staining with neutral red. In contrast, Vero CCL81 cells produced less clear plaques and was most easily quantitated by staining with neutral red 3 days postinoculation. On the individual plaque monolayers, SARS-CoV-2 infection of Vero E6 cells produced CPE with areas of cell clearance (Figure 2, panel C). In contrast, Vero CCL81 cells had areas of dead cells that had fused to form plaques, but the cells did not clear. Together, these results suggest that VeroE6 cells might be the best choice for amplification and quantification, but both Vero cell types support amplification and replication of SARS-CoV-2.
Thumbnail of Cell lines from patient with coronavirus disease, United States, 2020, susceptible to SARS coronavirus 2 (SARS-CoV-2). Cell lines were infected with a high multiplicity of infection (>5), washed after adsorption, and subsequently harvested 24 h postinfection for viral titer and protein lysates. A) Viral titer for SARS-CoV-2 quantitated by plaque assay on Vero E6 cells 2 days postinoculation. Infected cell protein lysates were probed by using Western blotting with B) rabbit polycl

Figure 3. Cell lines from patient with coronavirus disease, United States, 2020, susceptible to SARS coronavirus 2 (SARS-CoV-2). Cell lines were infected with a high multiplicity of infection (>5), washed after adsorption, and...
Because research has been initiated to study and respond to SARS-CoV-2, information about cell lines and types susceptible to infection is needed. Therefore, we examined the capacity of SARS-CoV-2 to infect and replicate in several common primate and human cell lines, including human adenocarcinoma cells (A549), human liver cells (HUH7.0), and human embryonic kidney cells (HEK-293T), in addition to Vero E6 and Vero CCL81 cells. We also examined an available big brown bat kidney cell line (EFK3B) for SARS-CoV-2 replication capacity. Each cell line was inoculated at high multiplicity of infection and examined 24 h postinfection (Figure 3, panel A). No CPE was observed in any of the cell lines except in Vero cells, which grew to >107 PFU at 24 h postinfection. In contrast, HUH7.0 and 293T cells showed only modest viral replication, and A549 cells were incompatible with SARS-CoV-2 infection. These results are consistent with previous susceptibility findings for SARS-CoV and suggest other common culture systems, including MDCK, HeLa, HEP-2, MRC-5 cells, and embryonated eggs, are unlikely to support SARS-CoV-2 replication (2022). In addition, SARS-CoV-2 did not replicate in bat EFK3B cells, which are susceptible to MERS-CoV. Together, the results indicate that SARS-CoV-2 maintains a similar profile to SARS-CoV in terms of susceptible cell lines.
Having established robust infection with SARS-CoV-2 in several cell types, we next evaluated the cross-reactivity of SARS-CoV antibodies against the SARS-CoV-2. Cell lysates from infected cell lines were probed for protein analysis; we found that polyclonal serum against the SARS-CoV spike protein and nucleocapsid proteins recognize SARS-CoV-2 (Figure 3, panels B, C). The nucleocapsid protein, which is highly conserved across the group 2B family, retains >90% amino acid identity between SARS-CoV and SARS-CoV-2. Consistent with the replication results (Figure 3, panel A), SARS-CoV-2 showed robust nucleocapsid protein in both Vero cell types, less protein in HUH7.0 and 293T cells, and minimal protein in A549 and EFK3B cells (Figure 3, panel B). The SARS-CoV spike protein antibody also recognized SARS-CoV-2 spike protein, indicating cross-reactivity (Figure 3, panel C). Consistent with SARS CoV, several cleaved and uncleaved forms of the SARS-CoV-2 spike protein were observed. The cleavage pattern of the SARS spike positive control from Calu3 cells, a respiratory cell line, varies slightly and could indicate differences between proteolytic cleavage of the spike proteins between the 2 viruses because of a predicted insertion of a furin cleavage site in SARS-CoV-2 (16). However, differences in cell type and conditions complicate this interpretation and indicate the need for further study in equivalent systems. Overall, the protein expression data from SARS-CoV nucleocapsid and spike protein antibodies recapitulate replication findings and indicate that SARS-CoV reagents can be used to characterize SARS-CoV-2 infection.
Thumbnail of Multistep growth curve for severe acute respiratory syndrome coronavirus 2 from patient with 2019 novel coronavirus disease, United States, 2020. Vero CCL81 (black) and HUH7.0 cells (green) were infected at a multiplicity of infection of 0.1, and cells (solid line) and supernatants (dashed line) were harvested and assayed for viral replication by using TCID50. Circles, Vero CCL81 cells; squares, Vero CCL81 supernatants; triangles, HUH7.0 cells; inverted triangles, HUH7.0 supernatant

Figure 4. Multistep growth curve for severe acute respiratory syndrome coronavirus 2 from patient with coronavirus disease, United States, 2020. Vero CCL81 (black) and HUH7.0 cells (green) were infected at a multiplicity of...
Finally, we evaluated the replication kinetics of SARS-CoV-2 in a multistep growth curve. In brief, we infected Vero CCL-81 and HUH7.0 cells with SARS-CoV-2 at a low multiplicity of infection (0.1) and evaluated viral replication every 6 h for 72 h postinoculation, with separate harvests in the cell-associated and supernatant compartments (Figure 4). Similar to SARS-CoV, SARS-CoV-2 replicated rapidly in Vero cells after an initial eclipse phase, achieving 105 TCID50/mL by 24 h postinfection and peaking at >106 TCID50/mL. We observed similar titers in cell-associated and supernatant compartments, which indicated efficient egress. Despite peak viral titers by 48 h postinoculation, major CPE was not observed until 60 h postinoculation and peaked at 72 h postinoculation, indicating that infected monolayers should be harvested before peak CPE is observed. Replication in HUH7.0 cells also increased quickly after an initial eclipse phase but plateaued by 24 h postinoculation in the intracellular compartment at 2 × 103 TCID50/mL and decreased after 66 h postinoculation. Virus was not detected in the supernatant of infected HUH7 cells until 36 h postinoculation and exhibited lower titers at all timepoints (Figure 4). Major CPE was never observed in HUH7.0 cells. These results are consistent with previous reports for SARS-CoV and MERS-CoV, which suggested similar replication dynamics between the zoonotic CoV strains (23,24).

Discussion​

We have deposited information on the SARS-CoV-2 USA-WA1/2020 viral strain described here into the Biodefense and Emerging Infections Research Resources Repository (https://www.beiresources.orgExternal Link) reagent resources (American Type Culture Collection, https://www.atcc.orgExternal Link) and the World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch (https://www.utmb.edu/wrcevaExternal Link), to serve as the SARS-CoV-2 reference strain for the United States. The SARS-CoV-2 fourth passage virus has been sequenced and maintains a nucleotide sequence identical to that of the original clinical strain from the United States. These deposits make this virus strain available to the domestic and international public health, academic, and pharmaceutical sectors for basic research, diagnostic development, antiviral testing, and vaccine development. We hope broad access will expedite countermeasure development and testing and enable a better understanding of the transmissibility and pathogenesis of this novel emerging virus.
The etiologic agent of an outbreak of pneumonia in Wuhan, China, was identified as severe acute respiratory syndrome coronavirus 2 in January 2020. A patient in the United States was given a diagnosis of infection with this virus by the state of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens from this patient and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into 2 virus repositories, making it broadly available to the public health and research communities. We hope that open access to this reagent will expedite development of medical countermeasures.
So they claim at least. Let's see their sauces.
A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as the source of a pneumonia outbreak in Wuhan, China, in late 2019 (1,2). The virus was found to be a member of the β coronavirus family, in the same species as SARS-CoV and SARS-related bat CoVs (3,4). Patterns of spread indicate that SARS-CoV-2 can be transmitted person-to-person, and may be more transmissible than SARS-CoV (57). The spike protein of coronaviruses mediates virus binding and cell entry. Initial characterization of SARS-CoV-2 spike indicates that it binds the same receptor as SARS-CoV angiotensin-converting enzyme, which is expressed in both upper and lower human respiratory tracts (8).
The citations in this article are so numerous I don't think I'll be able to cover them all. I will still do my best to link all of them as they appear.
There isn't much for me to say from my area of tenuous expertise. I have yet to discover a single rhetorical trick in this paper so far. Probably because it doesn't look good to a peer-review. There are assertions made such as SARS-CoV-2 being "identified" as the source of the pneumonia outbreak but they provide their sources. I'm unwilling to verify their veracity because they're equally dense articles and I'd be here for the rest of my life if I covered them all here. I might read them on my own time if I get the chance and I recommend you do so too, to help draw your own conclusions.
The unprecedented rapidity of spread of this outbreak represents a critical need for reference reagents. The public health community requires viral lysates to serve as diagnostic references, and the research community needs virus isolates to test antiviral compounds, develop new vaccines, and perform basic research. In this article, we describe isolation of SARS-CoV-2 from a patient who had coronavirus disease (COVID-19) in the United States and described its genomic sequence and replication characteristics. We have made the virus isolate available to the public health community by depositing it into 2 virus reagent repositories.
There is what I would construe as emotive language here regarding their "critical need" but it is a plea for research materials in the face of what is apparently a nascent pandemic. Like with Bailey's essay, Jennifer Harcourt et al. sees this as a matter of life and death so I can forgive a little urgency on both Bailey's and these people's parts.
According to this website, The Native Antigen Company, in their article Viral Lysates for Diagnostic Research:
Put simply, viral lysates are extracts of native virus from infected cells that have been treated to make them inert and safe for use. As a result, they contain a variety of native antigenic proteins and are able to elicit unique immune responses. Viral lysates are typically produced as follows:

1) Permissive cells are grown up in culture and infected with native live virus and cultured to allow sufficient viral replication.
2) The cell culture supernatant is added to a density gradient and ultracentrifuged until distinct bands are formed. Whole viruses have a different density to free proteins and cell debris, forming a recognisable band at a specific density.
3) The viruses are recovered from the gradient.
4) The viruses are then inactivated to make them safe for use. For enveloped viruses, this is done with detergent; For non-enveloped viruses, this is commonly done by heat, chemical or gamma radiation treatment.
A key characteristic of native viral lysates is that they contain all of the proteins that make up the virus, in the same structure as the virus is found in the host that it is infecting. This provides a source of antigens suitable for more complex or demanding immunoassays, where the total response to a virus rather than just a single antigen is being measured. This so-called ‘antigenic polyvalency’ of lysates allows the design of assays that are able to detect a wider range of antigens, and therefore increases sensitivity for detecting a pathogen. Lysate-derived antigen isoforms also show their utility in being able to stimulate immune responses that more closely match that of viruses in vivo.
In addition to diagnostic assays, typical applications include:

> Immunogens for raising antibodies. For example, to raise new monoclonal or polyclonal antibodies for the development of new assays or therapeutic reagents.
> As targets for studying the immune response to viral infection – either in ELISA or Western Blot (an ELISA will give an indication of an overall response, whilst Western Blots allow researchers to see which proteins the antibody response is directed against).
> As a source for purifying specific proteins from the total lysate.
So if I have this right, lysates are essentially a "safe" version of a virus in the state it would be in while infecting a body.

Methods​


Specimen Collection​

Virus isolation from patient samples was deemed not to be human subjects research by the National Center for Immunizations and Respiratory Diseases, Centers for Disease Control and Prevention (CDC) (research determination no. 0900f3eb81ab4b6e). Clinical specimens from a case-patient who had acquired COVID-19 during travel to China and who was identified in Washington, USA, were collected as described (1). Nasopharyngeal (NP) and oropharyngeal (OP) swab specimens were collected on day 3 postsymptom onset, placed in 2–3 mL of viral transport medium, used for molecular diagnosis, and frozen. Confirmed PCR-positive specimens were aliquoted and refrozen until virus isolation was initiated.
It's all very dry so there's not much for me to take apart from it. The citation here is one of the same ones featured in the last segment. Previously when seeing this sort of thing I thought it was redundant but I guess it makes sense if you're referring to different information from the same document.

Cell Culture, Limiting Dilution, and Virus Isolation​


We used Vero CCL-81 cells for isolation and initial passage. We cultured Vero E6, Vero CCL-81, HUH 7.0, 293T, A549, and EFKB3 cells in Dulbecco minimal essential medium (DMEM) supplemented with heat-inactivated fetal bovine serum (5% or 10%) and antibiotics/antimycotics (GIBCO, https://www.thermofisher.com). We used both NP and OP swab specimens for virus isolation. For isolation, limiting dilution, and passage 1 of the virus, we pipetted 50 μL of serum-free DMEM into columns 2–12 of a 96-well tissue culture plate, then pipetted 100 μL of clinical specimens into column 1 and serially diluted 2-fold across the plate. We then trypsinized and resuspended Vero cells in DMEM containing 10% fetal bovine serum, 2× penicillin/streptomycin, 2× antibiotics/antimycotics, and 2× amphotericin B at a concentration of 2.5 × 105 cells/mL. We added 100 μL of cell suspension directly to the clinical specimen dilutions and mixed gently by pipetting. We then grew the inoculated cultures in a humidified 37°C incubator in an atmosphere of 5% CO2 and observed for cytopathic effects (CPEs) daily. We used standard plaque assays for SARS-CoV-2, which were based on SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) protocols (9,10).
Citations 9 and 10 lead to more dry scientific papers with more science jargon I don't understand and leave me with nothing to commentate on. Really the entire Methods section is just describing stuff that they did and while the methodology bears some relevance to Bailey's essay I don't think I'll be able to find it in the vast maze of citations. I think I can safely skip this entire chapter without losing too much. Here is the link if you want to peruse it yourself, and I encourage you do.

Results​


A patient was identified with confirmed COVID-19 in Washington State on January 22, 2020. CPE was not observed in mock infected cells (Figure 1, panel A). Cycle threshold (Ct) values were 18–20 for NP specimens and 21–22 for OP specimens (1). The positive clinical specimens were aliquoted and refrozen inoculated into cell culture on January 22, 2020. We observed CPE 2 days postinoculation and harvested viral lysate on day 3 postinoculation (Figure 1, panels B, C). We used 50 μL of passage 1 viral lysates for nucleic acid extraction to confirm the presence of SARS-CoV-2 by using the CDC molecular diagnostic assay (1). The Ct values of 3 nucleic acid extractions were 16.0–17.1 for nucleocapsid portion 1, 15.9–17.1 for nucleocapsid portion 2, and 16.2–17.3 for nucleocapsid portion 3, which confirmed isolation of SARS-CoV-2 (Ct <40 is considered a positive result). We also tested extracts for 33 additional different respiratory pathogens by using the Fast Track 33 Assay. No other pathogens were detected. Identity was additionally supported by thin-section electron microscopy (Figure 1, panel D). We observed a morphology and morphogenesis characteristic of coronaviruses.
I remember CPEs being mentioned previously in Bailey's essay, let's see what Encyclopedia Britannica has to say about it.
cytopathic effect (CPE), structural changes in a host cell resulting from viral infection. CPE occurs when the infecting virus causes lysis (dissolution) of the host cell or when the cell dies without lysis because of its inability to reproduce.
With this added information let's go back and check on what Bailey said.
The history of virology reveals that the types of cells eventually selected for these experiments have been those that have a propensity to breakdown with the claim of virus-induced ‘cytopathic effects’ (CPEs), rather than those that are, “relatively easy to grow in the lab,” as Wiles claimed in her article.
Here we have Bailey mocking Miss Dangerhair from earlier for claiming that the cells used to "isolate" viruses are ones that can be easily grown in the lab.
That’s why when virologists want to isolate a virus from a sample they’ll take the sample or some part of it and add it to some cells – usually ones that are relatively easy to grow in the lab – and then look to see if the cells die and/or if there are any virus particles released into the liquid nutrient bath the cells are growing in.
It looks like quibbling to me. Who's to say that those cells aren't relatively easy to grow in the lab? This comes not long before Bailey makes his suspicions known:
Vero E6 monkey cells have long been favoured by virologists, supposedly due to their “suitability” to host many viruses, but suspiciously also, because the aneuploid kidney line is more susceptible to toxic insults from additional ingredients such as the ubiquitous nephrotoxic antibiotics and antifungals added to the culture mix.
Now with my newfound knowledge I see what he was claiming here. Bailey is suggesting that the CPEs visualized in these experiments are in fact not CPEs (i.e., evidence of viral infection) and that rather these breakdowns are the result of purificatory agents that damage the initial sample, causing the cells to break down.
Of course, Bailey doesn't provide proof that these cells are at all susceptible to the aforementioned "toxic insults" and instead carries on to both proclaim and snipe:
When one group attempted to culture SARS-CoV-2, they had no desired result with human adenocarcinoma cells (A549), human liver cells (HUH7.0), human embryonic kidney cells (HEK-293T), and a big brown bat kidney cell line (EFK3B), but then declared they had a “viral isolate” following the observation of CPEs in Vero E6 cells. As is typical, there seemed to be no sense of irony for them that the purported human respiratory virus cannot be shown to “infect” the relevant cell type, let alone the relevant species. And their experiments were once again invalidated by the absence of appropriate control cultures.
The citation in this segment leads back to the article I'm currently talking about, so the proof is in the pudding that I am currently eating. His claims have yet to be verified so I'll leave that until I'm finished with this thing. I also don't know what would constitute an appropriate control culture.
But taken as it is, this is evidence pointing toward a severe issue with confirmation bias in the isolation of SARS-CoV-2, but provides no proof of the alleged conspiracy, nor any proof that this problem extends to the entirety of the field of virology.
That out of the way I think I'm reaching the point where I'll have to divide this up again. I don't want to because from my cursory glances at Bailey's paper, the next segment is full of accusations apparently backed up with citations so I'll have a lot to sink my teeth into.
I'll leave you all with Figure 1 of the CDC's article.
20-0516-F1.jpg
Cytopathic effect caused by severe acute respiratory syndrome coronavirus 2 from patient with coronavirus disease, United States, 2020. A–C) Phase-contrast microscopy of Vero cell monolayers at 3 days postinoculation: A) Mock, B) nasopharyngeal specimen, C) oropharyngeal specimen. Original magnifications ×10). D) Electron microscopy of virus isolate showing extracellular spherical particles with cross-sections through the nucleocapsids (black dots). Arrow indicates a coronavirus virion budding from a cell. Scale bar indicates 200 nm.
 
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So this is still a thing right? Because that ream of text I'm not going to read would indicate you were, in fact, lying when you said this. Or you're just inconsistent. Either way...it hasn't stopped being hilariously ironic.:lol:
For someone who's no longer taking me seriously you seem to be taking up a lot of your time putting tophats on my posts and responding consistently whenever I quote you. If my existence upsets you so much there's a button you can press that hides my posts from view.
ignore.png

Or would acknowledging to yourself how much I annoy you mean that I "won"?
Besides, it's only inconsistent if you don't think about it. Mass Debates is for retards. I'm retarded. Where's the issue?
 
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So this is still a thing right? Because that ream of text I'm not going to read would indicate you were, in fact, lying when you said this. Or you're just inconsistent. Either way...it hasn't stopped being hilariously ironic.:lol:
Prove electro microscopes wrong, tryhard. Entire medical academia is against you and you only can sperg about semantics.

Have you you considered joining flat earthers, they could use your unparalleled intellect.
 
So this is still a thing right? Because that ream of text I'm not going to read would indicate you were, in fact, lying when you said this. Or you're just inconsistent. Either way...it hasn't stopped being hilariously ironic.:lol:
Sounds like someone's engaging in bad faith here.
Prove electro microscopes wrong, tryhard. Entire medical academia is against you and you only can sperg about semantics.

Have you you considered joining flat earthers, they could use your unparalleled intellect.
Even if he could prove electron microscopes are totally useless and always full of artifacts, he'd still be wrong about the non-existence of viruses since the pithovirus and pandoravirus (and several related orders of viruses) are over 1 micrometer long, which is easily visible on a lab-grade optical microscope. So it's on him to explain just what these are and why they kill certain species of amoeba when they come in contact.

But OP/his perpetually assmad boyfriend can't answer this question because it proves them wrong.
 
Even if he could prove electron microscopes are totally useless and always full of artifacts, he'd still be wrong about the non-existence of viruses since the pithovirus and pandoravirus (and several related orders of viruses) are over 1 micrometer long, which is easily visible on a lab-grade optical microscope. So it's on him to explain just what these are and why they kill certain species of amoeba when they come in contact.

But OP/his perpetually assmad boyfriend can't answer this question because it proves them wrong.
OP got his feelings hurt after he got banned in COVID thread, there are mountains of pictures, RNA breakdown of multiple virii and even man made viruses used in cancer treatments. I guess both of them dropped out of basic elementary biology.

I wonder how both of them explain why they get sick? Magic? Seeing flat earth logic being used on farms is baffling to say the least.
 
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