The Initial Isolation and Purification of the Rona – Part One

Activist Post
By Julie Beal

 

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Part 2

Some people reckon they can prove SARS-CoV-2 doesn’t exist by asking researchers around the world to provide them with records that show the virus has been isolated. The sense of victory grows with every reply that confirms ‘no such record exists’. But the game is rigged, because they will get the same reply every time, guaranteed. Really, they might as well give up now and just accept nobody can isolate a virus in the way they want it to be done.

Viruses are always cultured in cells in a lab, but toxic products are not used in the process, as explained in a previous article.

The activists’ request records, “describing the isolation of a SARS-COV-2 virus, directly from a sample taken from a diseased patient, where the patient sample was not first combined with any other source of genetic material (i.e. monkey kidney cells aka vero cells; liver cancer cells).” Their request then makes it clear they do not want to see records which employ the three main techniques used by virologists to obtain and characterize a virus, namely culturing, PCR tests, and genetic sequencing.

However, if we are ever going to find out more what is going on in the body, at the microscopic level of the cell, we’re going to have to consider use of these techniques because they are some of the best available. Rejecting the most common tools of the trade means we’re left in the dark about viruses or any other theory about what viruses ‘really’ are because nobody has come up with any better ideas!

Really, the only way to work out if anybody has ever isolated the rona is to look at the evidence yourself. Otherwise, you’re taking someone’s word for it. Having said that, the papers that describe the initial isolation and purification of the virus all sound like gobbledegook, so this article is like a primer for the one that comes after – part two will describe/translate the first three studies claiming to purify and sequence the SARS-CoV-2 virus in China. It’s not fun to read and there’s a lot of technical terms and techniques so the following information is intended to help you interpret their claims, as well as the claims of the no-virus theorists.

Isolation/Purification

The way virologists see it, isolation means removing material from a patient and keeping it going by putting it in a cell culture. They can also make it ‘extra isolated’ or ‘concentrated’ by purifying the sample, and by purifying the cell culture. Purification involves removing cellular debris and/or extracting the RNA or DNA. To help illustrate how these terms are used, here’s an example from the NIH patent for the pimped-up spike that’s in most of the ronavax:

Prefusion coronavirus spike proteins and their use‘ – “An ‘isolated’ biological component has been substantially separated or purified away from other biological components, such as other biological components in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA, RNA, and proteins. Proteins, peptides, nucleic acids, and viruses that have been ‘isolated’ include those purified by standard purification methods.”

But there’s no pandemic!

Exactly!!! That’s why we have to distinguish between the virus and the disease. Apparently, we’re all full of viruses which we don’t even notice, in the same way we don’t notice all the trillions of bacteria inside us. This is because viruses very rarely cause disease, and some even help us, so even if there’s a lack of a disease called covid, it doesn’t mean the virus is fake.

Whether covid exists or not is a separate matter, and for now we’re just trying to find out if the rona exists as a physical thing in people’s bodies, i.e. as a genetic construct, because we want to know if it was put there on purpose, and who might have done it. This is also why we have to review the evidence for viruses from cell cultures! If someone wanted to design and release a virus, they’d use a cell culture to propagate it, so the more we know about it, the better. Improving our understanding can help us argue our case more competently, such as being able to provide substantiated reasons for criticisms.

COMPARING METHODS OF ISOLATION AND PURIFICATION

The correct method according to Cowan, Fallon-Morell and Kaufman

The authors of the SOVI Declaration say a specimen taken directly from a patient should be purified by macerating, filtering and ultracentrifuging it. Afterwards, the “genetic makeup is characterized by extracting the genetic material directly from the purified particles and using genetic-sequencing techniques, such as Sanger sequencing…” The SOVI Declaration claims this has never been attempted with SARS-CoV-2 but it does so without making any reference to the studies published in China at the very beginning of the ronascam, when the ‘new disease’ was first declared. These are the studies which lay the foundations, and will be the subject of the second part of this article. Subsequent studies by other researchers were corroborating their findings. Many of them involved the isolation of viruses from patient samples which were then fully sequenced and had the same results (except for small mutations).[i]

The method used by Lanka et al

A paper co-authored by Stefan Lanka in the 1990s describes isolating a bacteriophage from marine algae. Bacteriophages are really tiny viruses that infect bacteria, and they’re cultured in a lab in a substrate that bacteria can live in. In order to purify their bacterial culture, Lanka and colleagues used the following method: “The particles were prepared from disrupted cells and purified by a procedure involving differential centrifugation, followed by PEG precipitation and centrifugation through a caesium chloride step gradient. Essentially all cellular debris … were removed by differential centrifugation.” The paper also includes photographs of the viral particles seen coming out of cells. For reasons unknown, Lanka disallows use of similar techniques for isolating or purifying viruses from human specimens using cell cultures.

Purification of viral particles

As mentioned above, samples and cell cultures are purified to isolate or concentrate viral particles. “Purification in a chemical context is the physical separation of a chemical substance of interest from foreign or contaminating substances. Pure results of a successful purification process are termed isolate.” Samples thought to contain viruses are usually purified using a centrifuge, which involves spinning the sample at high speed so the heavier stuff sinks to the bottom. This process is described in detail by Dr Osamu Nakagomi in ‘Fundamentals of Ultracentrifugal Virus Purification’:

“In recent years, in virus research, it has become a standard practice to purify and analyze genomes and identify viruses from samples using commercial kits. Since for the established viruses their genomes have already been known, virus identification is possible even in a mixed state. However, to carry out detailed investigation on the nature of viruses, it is first necessary to refine the virus particles in order to yield a high level of purified materials. When extracting virus genome using the classical method, the virus particles must first be purified. Then the virus genome extracted from the particles is examined.

Ultracentrifugation plays an important role in the process. … while studying new viruses, it becomes increasingly necessary to investigate whether or not the genome is present in the particle. In such cases, purification with an ultracentrifuge becomes a necessity. Information on the buoyant density, size and sedimentation coefficient (Svedberg value, S value), all of which are taken into consideration in ultracentrifugation, is in fact the fundamental aspect of virology which taken together are called the physiochemical properties of viruses.”

Most protocols involve centrifugation (spinning at really high speeds) “to remove the insoluble material (extracellular membranes, polysaccharides, and high molecular mass DNA)”, then further centrifugation to precipitate the RNA and form a pellet. Apparently, you need a high concentration of viral particles in order to purify them, because the very process of purification can rupture the particles, so the trick is to try and keep them intact, e.g. so they can be imaged using cryo-electron microscopy; “… higher amounts of infectious virus are recommended to recover enough sample for study, and to make it possible to recover a fairly complete virion proteome. Likewise, the accuracy of cryo-EM results depends on both the concentration and intactness of the purified virions. This requires a concentration step…”

Sucrose gradient purification of coronaviruses is described here.

Purification of Exosomes

To isolate and purify exosomes, density gradient centrifugation at lower speeds is used, “to remove cell fragments and large platelet-derived vesicles, followed by ultrafiltration and size-exclusion chromatography”. The repeated centrifugation of a sample containing exosomes is said to be, “more an enrichment of the sample than a purification”.

Ultracentrifugation-Based Exosome Isolation – You can separate exosomes from other sample components via differential centrifugation…. This method is often used in combination with sucrose density gradients or sucrose cushions to further purify exosomes according to their density. For this setup, you would centrifuge the pellet (containing the exosomes) at 100,000–200,000 x g for 120 min in a centrifuge containing a pre-constructed sucrose gradient medium, with decreasing sucrose density from bottom to top. Exosomes should float at sucrose densities ranging from 1.15 to 1.19 g/mL on continuous sucrose gradients.” The best way to purify exosomes is to use both methods but this “requires a large amount of starting material” (which is a problem when it comes to samples taken from patients!).

“…for rapid exosome enrichment, you may use nano-filtration concentrators with short periods of centrifugation.”

Extracting nucleic acids from the sample

Another way to characterize the components of a cell is to extract the nucleic acids from the sample or culture in question. This means removing either all of the DNA or all of the RNA. (See, for example, the ‘single step method of RNA isolation’)

DNA, RNA, and protein can be isolated from any biological material such as living or conserved tissues, cells, virus particles, or other samples for analytical or preparative purposes. RNA is separated from DNA after extraction with acidic solution… total RNA will remain in the upper aqueous phase of the whole mixture, while DNA and proteins remain in the interphase or lower organic phase.”

Extracting nucleic acids used to be a laborious process but now there are lots of specialist kits available, most of which involve, “repeated centrifugation steps, followed by removal of supernatants”. For example, “The RNeasy Plus Universal Mini Kit integrates fast, convenient RNA purification with effective elimination of genomic DNA. Optimized protocols enable purification of high-quality RNA from any type of tissue…” Thermo Fisher produce a viral RNA purification kit for use with biological fluids: “Successful viral analysis starts with the isolation of highly pure, concentrated viral RNA and DNA. Our advanced nucleic acid purification technologies for isolation of viral nucleic acids from biological (animal, insect, plant, fungal, and bacterial) and environmental (water, air, and food) samples give you confidence in your sensitive downstream real-time PCR and sequencing.” You can also use magnetic beads to extract DNA.

An Optimized Metagenomic Approach for Virome Detection of Clinical Pharyngeal Samples With Respiratory Infection’, by Liu et al (2020) compares the slightly different techniques available.

How Genomic Sequencing Changed The Game

Advances in genetic sequencing have led to various techniques becoming standardised in labs around the world, enabling scientists to sequence the genes of millions of organisms. Genomic sequencing has changed the game when it comes to characterizing the building-block identity of biological constructs, from things as small as viruses and bacteria, to things as large as humans and elephants. As explained in ‘Metagenomics: a path to understanding the gut microbiome’ by Yen and Johnson (2021), “The ability to sequence, assemble, and analyse whole genomes has sparked a genomic revolution that began with the completion of the human genome and continues today (Choudhury et al. 2020; Lander et al. 2001; Weinstock 2007). However, it is increasingly apparent that the human genome does not operate in isolation, rather it is part of a holobiont; a co-existing and co-evolving collection of host and microbial genomes that encompasses not just all three domains of life, but also viruses (Zilber-Rosenberg and Rosenberg 2008). Applying the same high-throughput sequencing technologies that revolutionised human genomics to the microorganisms “that literally share our body space” (Lederberg and McCray 2001) has resulted in a genomic revolution of its own.”

Shotgun sequencing can characterize entire microbial genomes (‘the metagenome’), all of which are interwoven with human individuals to form ‘holobionts’.

For microbiome studies, the next-generation sequencing (NGS) technology that has done most to bring about this paradigm shift is the massively parallel sequencing enabled by second-generation sequencing platforms. This has allowed massively parallel detection, quantification and, in the case of metagenomics, characterisation of thousands of microbial taxa within a single sample…. Subsequent arrival of third-generation technologies, such as nanopore sequencing … has coupled massively parallel sequencing with the ability to produce long reads (typically tens of thousands of bases per read). For microbiome research, long reads have meant a greater ability to identify the taxonomic origin of reads and hence better understand the composition of microbial communities. They have also improved the ability to assemble and annotate individual genes and genomes, leading to improved functional characterisation.”

The first bacterial genome to be fully sequenced was the H. influenza genome in 1995, and after that, whole-genome shotgun (WGS) sequencing really took off. This method involve cutting the bacterial DNA “into many small, easily sequenced pieces” and then using a computer “to align overlapping segments and thus assemble the entire genome.” These overlapping segments are called contigs, and although they’re made up of smaller pieces, they’re not small enough or ‘plain’ enough to just make up any old genome, as some researchers have been suggesting. In addition, genetic sequencing has to be performed with a computer because genomes are far too big and complicated to be able to work it out on a piece of paper, let alone fabricate entire genomes as “mental constructs”, as suggested by many of the same researchers. Anyhow, whole-genome shotgun sequencing is now “the method of choice for most sequencing projects large and small.”

Sanger sequencing, developed in the 1970s, used to be the most popular method, but shotgun sequencing allows researchers to analyse longer fragments. Overlapping DNA fragments are cloned and sequenced separately, and then assembled into one long contiguous sequence (or ‘contig’) in silico. This was achievable because the polymerase chain reaction (PCR) allowed them to produce the high concentrations of pure DNA species that are required for sequencing, and “… in recent years the Illumina sequencing platform has been the most successful, to the point of near monopoly”.

“Next-generation sequencing (NGS) is a relatively established technology in viral diagnostics”, including looking for new viruses. “Metagenomic sequencing is one of the commonly used NGS strategies for viral genome sequencing …. The main challenge for metagenomic sequencing in viral genome sequencing is enriching small amounts of viral particles from large host and bacterial genomes.” Samples are typically enriched before being sequenced, e.g. using filtration, digestion enzymes and centrifugation.

Metagenomic sequencing can also be used to analyse “the diversity, structure and potential functions of microbial communities in different environments”, such as samples of water or sediment, which can’t be cultivated. Researchers say that, “metagenomics can shed light into the diversity of viruses and their role in natural ecosystems.”

These microbial communities are part of the web of molecules that make up all living things, and form the terrain of which we are all a part. None of us can be isolated from the web of life so we need to work out how we all interact, and genetic sequencing is one of the ways we can clearly identify different types, and different effects. Analysing the microbes that inhabit and surround us could also help us understand some of the damage caused by vaccines. Be warned, though – the technocrats are already finding more ways to make money from microbes; “Put crudely, the idea is to use gut bugs as drugs.”

How PCR is used for genetic sequencing

As described on the Human Genome Project website,

“Polymerase chain reaction (PCR) is a laboratory technique used to amplify DNA sequences. The method involves using short DNA sequences called primers to select the portion of the genome to be amplified. The temperature of the sample is repeatedly raised and lowered to help a DNA replication enzyme copy the target DNA sequence. The technique can produce a billion copies of the target sequence in just a few hours.”

PCR has allowed geneticists to map the human genome as described in this quote from 1991:

“From both conceptual and practical perspectives, the polymerase chain reaction (PCR) represents a fundamental technology for genome mapping and sequencing. The availability of PCR has allowed definition of a technically credible form that the final composite map of the human genome will take…. Moreover, applications of PCR have provided efficient approaches for identifying, isolating, mapping, and sequencing DNA, many of which are amenable to automation. The versatility and power provided by PCR have encouraged its involvement in almost every aspect of human genome research, with new applications of PCR being developed on a continual basis.”

Making sense of genetic sequencing

It is not possible to use sequencing results to construct any virus you fancy.

The way Lanka and others make it sound, it’s as if scientists are combining letters of the alphabet together to come up with any story they like. However, it’s perhaps more like finding whole paragraphs or chapters, written in a specific language and presented in a particular format, but sometimes they end abruptly in the middle of a sentence and will only join with the other part of the sentence in the paragraph that follows. Another way to think of it is like a 3D jigsaw with complicated designs, colours and textures on each piece, which means they only fit together in a certain way.

The same methods are used to sequence the genomes of lots of different organisms so perhaps there’s a lot we can learn from the results of genetic tests. This is not to condone genetic engineering, gene therapy, or the manipulation of RT-PCR to create a fake pandemic based on ‘cases’, but merely to make the case for considering the evidence from the genetic sequencing of alleged viruses.

Stefan Lanka says scientists use ridiculously short sequences to construct the genome of a virus: “They always use very short pieces of nucleic acids, whose sequence consists of four molecules to determine them and call them sequences. From a multitude of millions of such specific, very short sequences, virologists mentally assemble a fictitious long genome strand with the help of complex computational and statistical methods.” (A human would create the programs to do this though! And the reason they have to use programs is because genetic sequencing would take too long to do with a pen and paper, and far too complex to assemble mentally!)

Cowan and Kaufman have made similar erroneous claims about sequences being extremely short, not covering the whole genome, and containing unknown genetic material. These claims have been echoed by yet more doctors, such as Dr Mark Bailey and Dr John Bevan-Smith, who claim, “What takes place is simply the shotgun sequencing of crude samples which contain genetic fragments of unknown provenance. Therefore, there is no evidence whatsoever, not even the vaguest guarantee, that the resulting in silico “genome” exists in nature or has anything to do with a ‘virus’.” (see, ‘The Covid-19 Fraud & War On Humanity’)

Notes:

[i] Slight changes here and there are normal for viruses and they’re classed as mutations or variants, and this is one of the reasons the genomes are fully sequenced (to keep track of mutations). Bear in mind this is not the same as confirming the mutations have different effects on people! Nor does it confirm the rona to be a risk. It’s just a read-out from the genome – the unique molecular fingerprint.

 

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