How to build a virus – a detailed lesson (very long)

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  • Sun, Aug 09, 2020 - 03:31pm

    #1
    Dr. Jurgen Mayer

    Dr. Jurgen Mayer

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    How to build a virus – a detailed lesson (very long)

As a virologist, I am interested in sharing science to help people better understand the topics that are being discussed on these forums. Just a warning that this post gets very technical, so do not continue if you are not interested in learning some biochemistry. Having a good grasp of how viruses are constructed will absolutely help you better understand the pandemic. My goal today is to bring you a little closer to being familiar with hCoV-2019.

Although I will be discussing many specific aspects of hCoV-2019, the vast majority is focused on general RNA viral formation.

A virus that has not infected a host cell is called a virion and that is the primary term we will be using today. We will create an example of a single virion entering a host domain (human body). Once the virion is in the host, the only intention is for the virion to replicate. It does not want to kill you or your cells, it needs your cells to stay alive. It simply wants to replicate and stay alive. We need to think of this virion like a computer program. It needs to be unpacked and installed in the operating system. Like with computer programs, these installation steps must occur in order. If we cancel/interrupt the installation at 80%, that would result in a failed installation. As with computer programs, the virion comes with an assembly manual. This manual provides very detailed instructions on how to assemble the new virion.

The assembly instructions are stored in an area of the data called an Open Reading Frame. If we draw a long line with a start and finish, this Open Reading Frame should be considered as the very start. Think of this as a table of contents but also IKEA furniture assembly instructions. These instructions are written using nucleic acids (NA). For DNA NA there are only four to work with; C (cytosine), G (guanine), A (adenine), T (thymine). For RNA, we simply replace Thymine with U (uracil). To recap, DNA has CGAT and RNA has CGAU. Do not worry about what these mean or what they do as that goes down a very complex biochemistry path. For now we will stick with our CGAU set. Nucleic acids are always read in collections of threes. We call this a codon. These combined sequences are called the genetic code. Here is a random string of RNA; CCCGAGUGACAT. We need to space that our three at a time. CCC GAG UGA CAT. Now we have four codons. Simple! We can generate a total of 64 possible codons (4x4x4=64). Simple as well.

Below is the actual beginning of the hCoV-2019 assembly. This is written as DNA and not RNA as it was taken from an infected human. As the virus is within the hosts’ cells we are reading the virus via the DNA genetic code of the human host.

GTAAGATGGA GAGCCTTGTC CCTGGTTTCA ACGAGAAAAC

To view this as RNA we replace any T listing with U, as previously explained.

GUAAGAUGGA GAGCCUUGUC CCUGGUUUCA ACGAGAAAACAC

This type of string is called a nucleotide. We are looking for the START of Open Reading Frame 1a. Later, we will also be dealing with ORF1b. When discussing ORF1 in general terms we will refer to that as ORF1ab. In order to begin the assembly, we have to press START. The specific codon for START is AUG. So we are looking for AUG in that code. That will tell us where the BEGINNING of ORF1a actually is! First, we will space that sequence out into codons to make it easier to read:

GUA-AGA-UGG-AGA-GCC-UUG-UCC-CUG-GUU-UCA-ACG-AGA-AAA-CAC

Ok, great. Can you locate the AUG codon in this nucleotide?? It’s missing!! But how is that possible? If we look back at the unspaced string, you can clearly find AUG, so we know that it exists, but once we separate into codons, you cannot find it?! What is actually happening is that the nucleic acids prior to START are basically junk, so it would misalign our codons! So we must ignore or delete them and begin spacing codons at AUG…

AUG-GAG-AGC-CUU-GUC-CCU-GGU-UUC-AAC-GAG-AAA-ACA-C (delete the last one for our example)

You are looking at the actual initial section of the Open Reading Frame of hCoV-2019 from within an infected human host from Wuhan, China sampled on December 28th, 2019! Ok, but why is that relevant? Well, viruses mutate and by reviewing the genetic code of patients we can track every single mutation the virus makes.

Now that we found AUG we have a clear start for our nucleotide chain. We are now assembling within ORF1a. Once the beginning of the assembly instructions is read it reaches its first “component” to assembly. This component is a protease. For the sake of this lesson, let’s just call a protease a recycling bin. Within ORF1a, we are going to create two proteases. Let’s just call one a recycling bin and one a trash bin. Remember the nucleic acids that we had to throw out in order to reach the START AUG codon? Toss those in this trash can (protease). Next, we need to learn to identify STOP codons. Think of this as more like a PAUSE. These are trickery and are always coded as either UAA, UGA, or UAG. Do we have any of those in our example nucleotide? Not yet. Examples of PAUSE/STOP would be like an old telegram “START. I am writing to tell you hello STOP I hope this news reaches you quickly STOP Write me back soon STOP. TERMINATE DICTATION”

Now we are going to go another level deeper with biochemistry. Each codon translates to a single amino acid. There are 20 amino acids and 64 possible codons. That means there are not enough amino acids to go around, the codons will have to share. STOP does not code for any amino acid. So when reviewing amino acid chains you would not know where the stops are unless you review the nucleotide chain. Make sure you understand that point. These STOPS would be invisible in the amino acid chain, that should make sense.

Without getting too technical, when amino acids are “bonded” together, there is a “glue” between them creating a chemical bond, we will ignore that bond for today but think of amino acids being glued together. A chain of amino acids would look like this O-O-O-O-O. As an example, the start of our ORF1a represented in amino acids is MESLV but think of this as M-E-S-L-V. Each of those represents an amino acid. M will always be from the START codon!

M= START

E= either GAA or GAG

S= either AGU or AGC

L= either CUU, CUC, CUA, CUG

V= either GUU, GUC, GUA, GUG

If we review our codons we have, AUG-GAG-AGC-CUU-GUC

We find a good example slightly further into the ORF1a of hCoV-2019:

AUG GCA CUU GUG GCU UAG UAG AAG UUG AAA AAG GCG UUU UGC CUC AAC UUG AAC AGC CCU AUG

This reads as START-A-L-V-A-PAUSEPAUSE-K-L-K-K-A-F-C-L-N-L-N-S-P-START. This is a good example, here we can see it start building a new component, has some pauses, then completes the assembly and moves onto another section in ORF1a!

 

Now we begin to assemble the second part of ORF1, which is ORF1b

This is a very important assembly as it contains the RNA-dependent RNA-polymerase or RdRp is one of the most crucial components of a virus. Think of the RdRp as your drivers’ license number. The government would use that number to quickly match you in a database, but that quick lookup could only provide them with limited information about you. RdRp is the commander or central helix of viral replication and transcription. As the RdRp drives the ship for viral replication, we cannot understate its significance. For reference, the RdRp of hCoV-2019 is 932 amino acids.

This is the actual RdRp sequence from a human patient in Wuhan, China in December 2019. This is a 932 polypeptide (amino acid) chain

SADAQSFLNGFAVVQPVLHRAAQALVLMSYTGLLTSTMIKLVLLNSKLIVVASKKRTKMTILILTLLRDTLSLTTNMKKQFIIYLRIVQLLLNMTSLSLETVTWYHIYHVNVLLNTQWQTSSMLGILMKVIVTHKKYLSHTIVVMMIISIKRTGMILKTQIYYAYTPTVNVYAKLCKQYNSVMPCEMLVLLVYHIIKISMVTGMISVISYKPRQVVEFLLILIIHCCLYPPGLLQSHMLTLTQSLTLSGICNMTSRKRGNSLTVILNIGIRHTTQIVLTVWMTDAFCIVQTLMFYSLQCSHLQVLDHEKYLLMVFHLFQLDTTSESVLYIIRMTYIALDLVLRNYLCMLLTLLCTLLLVIYYINALRAFRFIPSQVTNQPTFDLLICSLNELNLCGCHSAACLVHSRSIINNLLSLTGHELVYLLQAAYGFVRVAADHQHIVSSGCDRKVRWRALSLVSTRKHTSNSVCLFYRFATCSYVALETPWRRSYQRHVNILKMALVAKLKKAFCLNLNSPMCSSNVRMLELHLMVMLWLSWQNSKAFSTVVVVRHLVSLSLMWAKYQWLTARFFFVRTVIKELVAIVTAPISHLTATSLALILMKIFKKTGTLNIAVVLPVNSCVSLTEGHTLAMSITTSVALMATLLSALKTFHVLVKLHALCPNNWTLLTLRGVYTAAVNMSMKLLGTRNVLKRAMNCRHLLKLNWQRNLTPSMGNVQILYFPIPSRLFNQGLKRKSLMALWVEFDLSIQLRHQMNATKCAFQLSSVIIVVKLHGRRAILLKPLANFVALRILKKVPLLVVTYPKMLLLKFIVQHVTIQKDLSIVLPNTIMNLAKPFFVRVVALLPLEAVCSLMLVAITSVPIGFHVLALTVVTIQVLLEKVPKVLMTTFLKYSKKRKSTSILLVTLNLMKRSPLFWHLFLLPQVLLWKLKVWI

This sequence is missing any STOP commands. This is because we are looking at this from a cleaned-up view. In order to see what lies beneath, we will take the core nucleotide chain and convert it to amino acids so we can see which codons are issuing STOP and START, which gives us this sequence. Key: M means START (AUG) and – is stop (UAA, UGA, or UAG)

SADAQSFLNGFAVVQPVLHRAAQALVLMSYTGLLTSTMIKLVLLNSKLIVVASKKRTKMTILILTLLRDTLSLTTNMKKQFIIYLRIVQLLLNMTSLSLETVTWYHIYHVNVLLNTQWQTSSMLGILMKVIVTHKKYLSHTIVVMMIISIKRTGMILKTQIYYAYTPTVNVYAKLCKQYNSVMPCEMLVLLVYHIIKISMVTGMISVISYKPRQVVEFLLILIIHCCLYPPGLLQSHMLTLTQSLTLSGICNMTSRKRGNSLTVILNIGIRHTTQIVLTVWMTDAFCIVQTLMFYSLQCSHLQVLDHEKYLLMVFHLFQLDTTSESVLYIIRM-TYIALDLVLRNYLCMLLTLLCTLLLVIYYINALRAFRFIPSQVTNQPTFDLLICSLNELNLCGCHSAACLVHSRSIINNLLSLTGHELVYLLQAAYGFVRVAADHQHIVSSGCDRKVRWRALSLVSTRKHTSNSVCLFYRFATCSYVALETPWRRSYQRHVNILKMALVAKLKKAFCLNLNSPMCSSNVRMLELHLMVMLWLSWQNSKAFSTVVVVRHLVSLSLMWAKYQWLTARFFFVRTVIKELVAIVTAPISHLTATSLALILMKIFKKTGTLNIAVVLPVNSCVSLTEGHTLAMSITTSVALMATLLSALKTFHVLVKLHALCPNNWTLLTLRGVYTAAVNMSMKLLGTRNVLKRAMNCRHLLKLNWQRNLTPSMGNVQILYFPIPSRLFNQGLKRKSLMALWVEFDLSIQLRHQMNATKCAFQLSSVIIVVKLHGRRAILLKPLANFVALRILKKVPLLVVTYPKMLLLKFIVQHVTIQKDLSIVLPNTIMNLAKPFFVRVVALLPLEAVCSLMLVAITSVPIGFHVLALTVVTIQVLLEKVPKVLMTTFLKYSKKRKSTSILLVTLNLMKRSPLFWHLFLLPQVLLWKLKVWI

This RdRp contains 41 STOPS and 47 STOPS. This tells us that this RdRp section is VERY busy with assembly and highlights how important this portion is to a virus! The reason the RdRp does not begin with an (M) START codon is that this is an assembly within a polyprotein construct (ORF1). As viruses mutate we will see changes occur throughout the virus but specifically within the RdRp as the virus attempts to evolve in order to survive within its host. The mutations with an RdRp are almost entirely related to fine-tuning the replication process. An RdRp is such a key feature of a virus that is the pre-dominant testing point for PCR testing. When a patient is “tested” for “COVID-19” the PCR testing device is looking to match any RdRp chain found within your DNA to a sample like the one above. As the example above is the actual RdRp from hCoV-2019, the PCR test is trying to seek a positive match. But what is the testing strength? The RdRp from this 2019 Wuhan sample is exceptionally different from a July 2020 sample from South Africa (70-year-old female patient). In this example, the source RdRp is only 73.67% similar to what we see in July 2020. Given that an RdRp will greatly mutate over time, we should generally question the accuracy of PCR testing when it comes to attempting to match a test to a certain virus.

Once the RdRp assembly is completed in ORF1b, it continues to build some other, less important components. Then builds the (S) Spike protein. This protein is very important and contains a lot of complex information about how to potentially dock to host cell receptors. The most important section of the Spike protein is called the Receptor Binding Domain. This is an area of the protein that opens and closes in order to lock the docking process. The Spike protein is so complex that it requires its own write-up. People often focus on the (S) Spike protein of a virus and ignore most everything else and that is a mistake. Always remember that it is the ORF that is providing the assembly instructions for the Spike protein. When mutations occur within a Spike, or any other protein, they are typically occurring through the ORF and passed to the protein during assembly.

After the Spike is completed, next is the assembly of the (E) envelope protein. In the case of hCoV-2019, this is a glycoprotein covering that will act as the outer shield of the virus. Why is that relevant? Let’s look at influenza. This virus also creates an outer shield but is a lipid bilayer. Think of this outer shell is soft plastic. You think you may have caught influenza. In this example it is infecting your sinus, so you initially get congestion. If you use a nasal flush solution you can flush most of the virus particles down into your stomach. Once there, your stomach acids will actually destroy the virus. The lipid bilayer of influenza is not enough protection from stomach acids. This is true of many viruses. hCoV-2019 and others instead have a glycoprotein surrounding their envelope. So we take that soft plastic shell and now cover that with a firm plastic. Stomach acids cannot penetrate this firm plastic, which means very bad things. If you become infected with an enveloped glycoprotein virus in your sinus and attempt to “flush” the virus down to your stomach, what happens is that the virus will simply attempt to infect the cells in your stomach leading to various medical issues.

After this assembly is finished, next is the membrane protein. Compare the virus with an avocado. The outer-most shell is the envelope. Then imagine an inner shell that is the “construct” for which the shell is built-upon. This area is both the membrane and the matrix. Outside of the shell we are going to attach a bunch of spike receptors. The “meat” inside of the avocado is the capsid (explained below) and the pit is the RNA virion. This is the actual RNA payload. Once docking to a cell happens the virus pushes its RNA payload up through the avocado, through the docked spike and into the host cell. Avocado virology at its finest.

Next is the assembly of the (N) nucleocapsid protein. Its main function is to protect and encapsidate (enclosure of a virus particle) the viral RNA forming viral RNP complex. This “coat” is protecting the core (pit) payload.

Aside from these primary proteins that you have learned about during assembly, RdRp, S, E, M, N, there are also non-structural proteins or NSPs created from the ORF to serve as helpers for various functions. Think of these as ancillary cast members in a movie. They are there, they are assisting, but they are not important.

Although ORF1ab is of huge importance to the virus, it is not the only ORF being read for assembly. These are also ancillary but certain mutations in those ORFs could have impacts on the virus.

 

As each component is assembled, the polypeptide chain of amino acids is “bonded” together with a “glue” residue, and the component is then “folded” or converted into a ribbon type structure. This completed form is called a protein. Once all of these various proteins are assembled and the virus is ready to travel, this is called a mature virus, meaning that it is fully developed/assembled.

 

You should now be able to mostly understand this beautiful picture and see a visual in your head of how this assembly process is happening in order to create this maturation assembly.

 

Here is a more detailed breakdown of hCoV-2019. Everything we learned today is related in this one amazing photo.

 

If you bothered reading all of this, hopefully, this lesson has been very helpful for you. As you begin to understand the science of the virus, it brings you closer to the virus. Suddenly it is not as foreign or confusing.

  • Sun, Aug 09, 2020 - 08:26pm

    #2
    Grayman

    Grayman

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    How to build a virus – awesome explanation

Dr Mayer,

You have a gift for teaching. Thanks for that.

Grayman

  • Mon, Aug 10, 2020 - 06:27pm

    #3
    Island girl

    Island girl

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    How to build a virus – a detailed lesson (very long)

This  just published by Luc Montaigner:

https://zenodo.org/record/3975578

 

 

  • Tue, Aug 11, 2020 - 02:27am

    #5
    Mpup

    Mpup

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    How to build a virus – a detailed lesson (very long)

IG (Inspector General) 🙂   If one downloads the full text of the article (47 pages)  https://zenodo.org/record/3975578/files/678-Article%20Text-4089-3-10-20200806%20%281%29.pdf?download=1  it appears to confirm what Dr. Mayer says about the inadequacy of PCR testing because of mutations.  The full text will no doubt have more meaning to Dr. Mayer and others with a better understanding of virology.

This article appears to further confirm that Covid-19 is a lab created, manipulated virus.  They have built a monster.  From the conclusions on page 44 of the article:

4. CONCLUSIONS
1) 18 RNA fragments of homology equal or more than 80% with human or simian retroviruses have been
found in the COVID_19 genome.
2) These fragments are 18 to 30 nucleotides long and therefore have the potential to modify the gene
expression of Covid19. We have named them external Informative Elements or EIE.
3) These EIE are not dispersed randomly, but are concentrated in a small part of the COVID_19 genome.
4) Among this part, a 225-nucleotide long region is unique to COVID_19 and Bat RaTG13 and can
discriminate and formally distinguish these 2 genomes.
5) In the decreasing slope of the epidemic, this 225 bases area and the 1770 bases Spike region, exhibits
an abnormally high rate of mutations/deletions (cases of 44 patients from WA Seattle state, original
epicenter in USA).
6) In the comparative analysis of both SPIKES genes of COVID_19 and Bat RaTG13, we note two abnormal
facts:
• The insertion of 4 contiguous PRRA amino acids in the middle of SPIKE (then we show that this site was
already an optimal cleavage site BEFORE this insertion).
• An abnormal ratio of synonymous codons / non synonymous codons in the second half of SPIKE.
Finally we show the insertion in this 1770 bases SPIKE region of a significant EIE from Plasmodium Yoelii and
of a possible HIV1 EIE with a crucial Spike mutation.
Through the 14 facts relating to each of the 14 paragraphs of this article, everything converges towards possible
laboratory manipulations (End Note below) which contributed to modifications of the genome of COVID_19, but also,
very probably much older SARS, with perhaps this double objective of vaccine design and of “gain of function” in
terms of penetration of this virus into the cell.
This analysis, made in silico, is dedicated to the real authors of Coronavirus COVID_19. It belongs only to them
to describe their own experiments and why it turned into a world disaster: 650 000 lives (on 26 July 2020), more
than those taken by the two atomic bombs of Hiroshima and Nagasaki. We, the survivors, should take lessons from
this serious alert for the future of humanity. We urge our colleagues scientists and medical doctors to respect ethical
rules as expressed by Hipocrates oath: do not harm, never and never !
End Note: Why could COVID-19 come from Laboratory manipulations?
The following 4 proofs concern differences with respect to SARS either common to COVID-19 and bat RaTG13,
or facts radically differentiating these 2 sequences of which it is claimed that the first (COVID-19) comes from a
natural evolution of the second (bat RaTG13). We have ranked these 4 proofs in ascending order of importance
according to our point of view.
1) Four EIE formally distinguishes COVID-19 and bat RaTG13 genomes from all other SARS or bats genomes.
However, their level of HIV/SIV homologies appears much more affirmed for COVID-19 than for bat
RaTG13, as if these EIE fragments had recently been “re-injected” into the COVID-19 genome. ==> see &
7, (figures 4 and 5).
2) natural deletions (USA WA Seattle state) apply in priority to EIE inserts (HIV Kenya etc ..). ==> see full
Part III and Figure 12 in §13.
3) Synonymous codons mutations within the 1770 bases region of the Spike, which simulate a natural
evolution of bat RaTG13 towards COVID-19 while maintaining the optimality obtained in amino acid
values, probably from “gain of function” Laboratory experiments (optimality common to both RNA
sequences COVID-19 and bat RaTG13) ==> see Figure 10 in & 11 and Figure 11 in §12.
4) “PRRA” amino acids was inserted exactly on the Spike location already theoretically optimal on both
COVID-19 and RATG13 (of which it constitutes the main difference). ==> see Figure 13 in & 14.

edit to add from the conclusions:  “This analysis, made in silico, is dedicated to the real authors of Coronavirus COVID_19. It belongs only to them
to describe their own experiments and why it turned into a world disaster”

Dedicated to the real authors? Surely you jest.  And we are to think they will describe their own experiments?  The fact that they have remained silent to date with hundreds of thousands of dead tells us they have no conscience.  They have committed crimes against humanity and should be held accountable.  The world cannot give these people regardless of nationality, or the CCP a pass.   There are a select few who know with certainty ground zero,  loose lips sink ships.

In thinking themselves wise, they became fools”

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