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A series of posts about virology

Thu Apr 02, 2020 4:57 pm

A long time ago in a galaxy far, far away, I was a molecular virologist. It was only for a year and I worked on a kind of virus called a "baceriophage," which infects baceria, but in the process, I learned a lot about virology.

I started writing a series of posts on Facebook about virology because...well...most of us have nothing better to be doing right now (leaving aside the fact that I'm...you know...a doctor). And so I've decided to share those posts here.

In order to follow this series of posts, you will need to have taken 9th-grade Biology. So here we go:

Some basics of Biochemistry

There are four main classes of biomolecules.

1) Lipids (fats): These store energy, but also (and more importantly) they also are the basic fabric of the cell membrane and viral envelope. These membranes are held together by the same forces that hold soap bubbles together.

2) Nucleic acids: There are two kinds of nucleic acids. DNA is a long-term information storage system. DNA is highly stable and it is the main information storage system of all living things. The second kind is RNA. Within living organizms, RNA serves a number of functions relating to a) information transfer (carries copies of genes out of the nucleus to the ribosome to be translated into proteins), protein production (ribosomes, tRNA), and a few other miscellaneous regulatory functions. Many viruses, including coronaviruses, use RNA as their genetic material.

3) Sugars: energy storage, also perform some functions with respect to protein-protein interactions. Not going to be a major part of this discussion.

4) Proteins: Proteins are strings of amino acids. There are 20 amino acids and the sequence of these amino acids in each kind of protein is determined by the genetic sequence. Some amino acids are positively-charged in water. Some are negatively charged in water. Some are greasy and some are like alcohol and like to dissolve in water. When the protein is being extruded from the ribosome, it starts to fold up. The positive amino acids find negative ones, the greasy ones congregate towards the center of the protein, and the alcohol-like ones go to the outside. The sequence of amino acids defines a very specific shape (and therefore function) that the protein will form and every single protein of a given type is identical.

What do proteins do? Well, almost everything. They are like little machines that run chemical reactions. They generate energy and they use energy. They can serve as signals both inside and outside the cell. They can join together to form structures that define the cell's shape. They move things. Antibodies are proteins. Insulin is a protein. The actin and myosin in your muscles are proteins. The various enzymes that maintain, read, and replicate your DNA are proteins. And an important thing to understand for this discussion is that **some proteins bind very specifically to certain other proteins.**
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Re: A series of posts about virology

Thu Apr 02, 2020 5:03 pm

and....? looking forward to this!
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Re: A series of posts about virology

Thu Apr 02, 2020 5:08 pm

Definitions and classifications of life on Earth and the relationship of viruss to life

Definitions
"Life" is a curious term. We all think we know what it is. Most of us agree that a rock is not alive, but a tree is. There are some things that meet some of the criteria for life. Fire uses energy, it reproduces, and it can even adapt. But fire is not an object. It carries no information, has no cells, and spontaneously forms. So let us go over some basic requirements for life on Earth. Some of these aren't mentioned in the textbooks because every biologist yearns to discover extraterrestrial life, but for the purposes of this discussion, I'm going to stick with life on earth.

*Life reproduces *AND* it must come from other life (notwithstanding the origin of life on Earth). It does not spring spontaneously out of nowhere.

*Life contains information in the form of DNA. But also, it contains information in the form of the various proteins and other molecules around the DNA.

*Life can adapt to its environment, within certain limits.

*Life uses and metabolizes energy.

*Life is made of cells.

*Life has a clearly defined "inside" and "outside." And when the boundary between "inside" and "outside" is sufficiently disrupted, it stops being life.

*Life strives to keep its internal conditions as consistent as possible, which is to say that life tries to maintain "homeostasis." Although there may be some significant variations on this (sporulation).

We will see that viruses meet some of these requirements, but not others.

Classifications
Broadly, life can be divided into two large groups, the eukaryotes and prokaryotes.

Prokaryotes are cells (they are all single-cell organisms) with no nucleus, and no internal structures that are bound by membranes. So no endoplasmic reticulum (remember that from 9th grade?), no mitochondria, and no nucleus.

Eukaryotes are organisms that are made of one or more cells that *do* have membrane-bound organelles, including a nucleus. Eukaryotes can be single cells (paramecium, amoeba, algae) or multicellular (oak trees, elephants, mushrooms). We humans are, of course, eukaryotes.

Viruses

A virus consists of a small bit of information. Notice that I did not specify that a virus need be surrounded by anything because for a good portion of its reproductive cycle, it is not. However, viruses are usually depicted as a virus "particle," which is also called a "virion." I will use these terms interchangeably. Here is a space-filling model and a schematic of an adenovirus particle (I didn't choose a coronavirus mostly out of spite).

Image

So why aren't they alive? Well, they aren't cells, many of them don't even have a membrane, they don't metabolize energy (rather, their components use the cell's energy that has already been metabolized) and as a normal part of their reproductive cycle, they completely disassemble.

But let's explore this with an argument. We'll go back to the bacteria and one specific bacterium, Escherichia coli, which most of us think as causing disease, but it actually lives quite happily in all of our bowels and usually doesn't cause trouble. It's also a major "model organism" for biologists because it's easy to grow and it's a simple organism that is easy to manipulate. Much of our original understanding of genetics comes from E. coli.

E. coli, like many other bacteria, can contain "plasmids." While E. coli's one chromosome is circular and contains all the genes that the bacterium needs, plasmids are small circles that contain only a few genes. They're generally a few thousand base pairs in length, about the size of a virus. They're just loops of DNA and I think it's safe to argue that they are not alive, even though they can reproduce (divide) and even manage to get transferred from one cell to the next.

Plasmid F was one of the first plasmids discovered in E. coli. I put a map of it in the images. It's called F for "fertility." When a bacterial cell contains plasmid F, the genes on the plasmid will code for a series of proteins that make a "sex pilus." Yup, it's actually called that. Here's a map of plasmid F:

Image

The sex pilus sticks out of the donor cell (the cell that has plasmid F) and that cell floats around until it finds another cell. The sex pilus, which is made of proteins, binds very specifically to a protein on the surface of the recipient cell (remember I said that proteins can bind to each other very specifically) and then a copy of plasmid F is transferred from donor to recipient and now both cells have plasmid F. Here's a transmission electron micrograph of what this "conjugation" event looks like with the sex pilus as the long structure connecting the two cells.

Image

Plasmids like F don't kill the cells that they "infect." They most certainly don't "want" to. (I'm going to personify biological systems a lot here because that's how biologists talk, but remember, even the majority of living things aren't conscious) And they sometimes carry useful genes, like antibiotic resistance genes. This is an evolutionary tactic that helps keep cells that have F competitive with their "uninfected" brethren. So F is not alive, and it's just a collection of information that allows it to be transferred from cell to cell.

So imagine that instead of forming a sex pilus and being threaded through from one cell to the next that the sex pilus completely filled with a copy of plasmid F and then broke off the donor cell and floated around until it found a recipient cell and then got transferred in that way. Well, plasmid F would have gone from a plasmid, which isn't alive, to a virus. And it's still not alive.

Well, other bits of genetic information also came across this bag of tricks, probably billions of years ago and they *did* make that jump and they became viruses. The viruses that infect bacteria are called bacteriophages. Well, when your existence no longer depends on keeping your host alive, you can try some different strategies. Some bacteriophages, like bacteriophage T4 (pictured...you recognize it), kill all the cells they infect. Others, like bacteriophage lambda, might integrate themselves into the host cell's genome and only break out and kill some of the cells under certain conditions. Others still, perform "chronic" infections in which phage particles are released over a long period of time without disrupting the host cell.

Image

OK, so what about viruses that infect eukaryotes? Well, the general principles are the same. And so in the next section, I am going to cover the virus reproductive cycle.
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Re: A series of posts about virology

Thu Apr 02, 2020 5:21 pm

Viral Replication

An overview of biological membranes

Before I begin, I need to cover some remedial biochemistry as it pertains to the cell membrane. All cells are surrounded by a cell membrane, harkening back to my point that all life has an "inside" and an "outside" and that any major disruption of this boundary is fatal.

The membrane is made up of a class of molecules called "phospholipids." Here's a diagram (both stick and space-filling) of one. A phospholipid belongs to a class of molecules called "amphipathic" molecules. That is to say that one end of the phospholipid (the end at the top of the image with the phosphate and ammonium components) is very soluble in water. The other end of the phospholipid is greasy and doesn't like to dissolve in water.

Image

When you take a bunch of phospholipids and drop them in water, they will spontaneously orient themselves such that the greasy, fatty ends of the molecules find each other and the charged, water-soluble ends of the molecules stick out into the water. This forms a "phospholipid bilayer" (pictured), which is the fundamental fabric of a cell membrane. Water can pass through, and some small molecules like alcohol can pass through (if there is too much alcohol, it messes with the greasy/not greasy interactions and the whole structure falls apart...more about that later). Not only that, but membranes can pinch off and form small circular "bubbles" of membrane called "vesicles" and vesicles can fuse into larger membranes. That's going to become important later in this series.

Image

Larger molecules like sugars and DNA cannot pass through, nor can charged ions like potassium, sodium, or chloride. So in order to allow these things in and out of the cell, proteins are embedded in the cell membrane (pictured). These proteins serve a number of functions. Some of them allow certain substances such as ions and sugars and even entire proteins in and out of the cell. Others transfer information in and out of the cell. You can see that many of them are free to diffuse laterally within the membrane, while others are attached to underlying structural elements. Some of them also attach to external structures outside the cell to help hold it in place.

Image

Remember, that some proteins bind *very specifically* to other proteins. When a protein on the surface of a cell binds specifically to another protein (or a small molecule like a neurotransmitter), we call that protein a "receptor" and that which binds to it is its "ligand" (ligg-und). Although just like there is no up and down in space, sometimes the question of which is the receptor and what is the ligand can get a bit...circular.

Anyway, that's what you need to understand about membranes because that's important to understanding viral binding and entry. Now, let's move on to the viral replication cycle.

Viral Replication Cycle

I will split the cycle into four phases.

1) Encounter and binding
2) Cell entry
3) Intracellular reproduction and/or latency
4) Egress

Encounter and Binding

All chemical reactions and indeed all biological processes take place in water. This may not be obvious when we see a bird flying overhead or a desert flower. Certainly we humans do not live in the water, but humans are 60-70% water by mass. The desert flower contains water. A loaf of bread contains about a glass of water.

Viruses may float through the air (usually in a tiny droplet of water), or they may reside on a surface (again often in a drop or thin layer of water), or we may find them in a body of water, be it a puddle, a river, or an ocean. Water is seldom pure. Some viruses are excreted in the stool and spread by a process called "feco-oral" transmission in which a microscopic speck of feces winds up on your lunch and you eat it (yeah, gross) and then it infects you.

But when a virus encounters a cell, it does so in the water. That water might be the moist mucous membranes of your nose or mouth or GI tract or the surface of your eye, or even your genital tract.

Viruses, and just about anything else suspended in water, will tend to float around at random unless some other force (buoyancy, current, intentional propulsion) acts on it. Viruses are usually not affected by these forces other than current, but as they float around at random, they will encounter the surface of a cell and bind to it.

The surface of a virus particle will have a number of proteins sticking out of it that serve as ligands for a receptor on the surface of a cell. The choice of surface receptor for a virus will determine what kinds of cells it can infect.

Bacteriophage lambda has a long tail with fibers on the end (pictured) that binds to a bacterial surface protein called LamB, which usually serves to move sugars in and out of the cell. Humans do not have this protein, and so we could swim in a lake of bacteriophage lambda and it would be unable to infect us.

Image

HIV binds to a receptor called CD4, which is primarily found on a kind of immune cell called a CD4+ T cell, which is why it kills these cells and causes AIDS (it also needs a second receptor, or a co-receptor called CCR5 and people with mutations in CCR5 can be very resistant to HIV infection and/or progression to AIDS).

And so, while I have avoided discussing the SARS Coronavirus 2 (SARS-CoV-2, AKA the Wuhan Coronavirus) out of spite, it is worth discussing it now. This coronavirus particle is covered in a "halo" or "corona" of proteins called "spike proteins" (that look nothing like spikes, more like clubs) and they bind to a surface receptor called ACE2, or Angiotensin-Converting Enzyme 2, a protein involved in the regulation of blood pressures. Those "spike" proteins are depicted in red on this now very familiar model of the virus particle pictured. The detailed intermolecular interactions of the spike protein with the ACE2 receptor is shown in this image obtained by a technique called cryoelectron microscopy. (1) (For those of you who are bewildered by that image, just enjoy the pretty picture). Now, not all coronaviruses use this receptor, but SARS-CoV and SARS-CoV-2 both did.

Image

That choice of receptor may be of clinical relevance. The ACE-2 receptor is found on cells all over the body including the kidney, the heart, the testis, etc. But in the lung, there is very little of it except on one specific kind of cell called a Type 2 Alveolar Cell (AT2). These cells are found in the alveoli (the microscopic air sacs where gas exchange between the air and blood occurs) and they secrete a kind of chemical called "pulmonary surfactant." This chemical keeps the alveoli from collapsing under the surface tension of the water that coats its interior. And now that I mention that, it might become obvious to you how this feature of this virus can lead to a disease in which alveoli are prone to collapsing under their own surface tension and cause respiratory distress.

(1)https://science.sciencemag.org/content/367/6485/1444.full
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Re: A series of posts about virology

Thu Apr 02, 2020 5:31 pm

Viral Entry

When a virus contacts a cell, the next step is for the virus to enter the cell. Remember that in many cases, the entire virus particle does not enter the cell, but rather only its contents.

For a bacteriophage like T4, the viral DNA is injected directly into the cell by a structure that looks just like a molecular needle (pictured). For bacteriophage lambda, it is threaded in through an existing protein that usually transfers sugars. (also pictured). Bacterial cells usually have a rigid cell wall, so such brutal mechanisms are necessary. But for animal cells, there are no cell walls and so there are multiple ways in which viruses might enter the cell.

Bacteriophage T4 DNA injection
Image

Bacteriophage lambda DNA injection
Image

I'll go over some examples, finishing with SARS-CoV-2.

For Norovirus, a virus that causes vomiting and diarrhea and is infamous for breaking out on cruiseships, it encounters the host through feco-oral transmission (a microscopic particle of poop on your food). In the intestines, bile salts attach to the outside of the virus particle, which does not have an envelope (membrane). The particle itself then binds to a surface receptor (which has not yet been identified in humans but is suspected to be the blood group antigen by some researchers) and that causes the membrane to pucker inwards and pinch off into a small bubble called a vesicle with the virus particle inside of it. A protein in the particle called Viral Protein 2 (vp2) drills through this little vesicle and releases the viral RNA into the cell. (Pictured) (1)

Image

HIV binds to two proteins on the surface of the T-cells it infects, CD4 and CCR5. A protein on the envelope then finds a partner and brings the two membranes into close proximity so that the membranes fuse. This allows the nucleocapsid (the core of the virus) to enter the cell, where it disassembles and begins the replication cycle.(pictured)

Image

Influenza has two kinds of proteins on the surface of its envelope. The first is called neuraminidase and it breaks down the slimy, sugary coating over mucous membranes in the respiratory tract to allow the virion to approach the cell (drugs like oseltamivir ["TAMIFLU"] inhibit this enzyme) and the second is called hemagglutinin. Hemagglutinin binds to a receptor on the surface and is taken again into a vesicle called an endosome. The endosome becomes acidic (which is a defensive feature), but this acidification actually activates the hemagglutinin on the surface of the virus particle, which makes the envelope of the virus particle fuse with the membrane of the endosome. This releases the proteins and RNA inside the virus particle into the cell where they can set up an infection.

SARS-CoV-2 uses a particular method of entering its target cells. First, a few of spike proteins (perhaps as few as 2 or 3) binds to the surface receptor ACE2. However, this binding alone is not enough to cause the cell to enter. But now that the virus is latched onto the cell, a second protein called TMPRSS2 comes along. TMPRSS2 is a protease, a protein that cuts up other proteins. TMPRSS2 starts cutting other unbound spike proteins, but rather than destroying them, the spike proteins are assembled in just such a way that the action of TMPRSS2 actually uncovers a second layer of machinery inside the spike. This machinery fuses the membrane of the virus particle with the cell membrane (Sometimes this happens inside an endosome, which requires acidification of the endosome, which is how hydroxychloroquine is theorized to work...if it does) and releases the contents of the particle into the cell.(2)

I will mention one more method of viral entry. Some viruses, such as respiratory syncytial virus and indeed our friend SARS-CoV-2 can cause cells to fuse directly together to form a single giant cell with two or more nuclei. This fused cell is called a syncytium ("sin-SISH-um"). Some syncytia are normal (muscle fibers are syncytia), but in this case, the virus is forcing the formation of a syncytium so that it can pass directly from one cell to the next without having to form a virus particle and exit the cell.

The replication cycle inside the cell is next.

(1)https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6630345/
(2)https://www.cell.com/cell/fulltext/S0092-8674(20)30229-4…
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Re: A series of posts about virology

Thu Apr 02, 2020 5:36 pm

Intracellular phase, Latency, and Egress

Latency

I'm only going to briefly touch on latency because the virus that has everyone's attention right now doesn't exhibit this behavior.

Latency is a term for when a virus infects a cell and then hides its genetic material there. During this time, any genes expressed from the latent viral genetic material are expressed at a very low level (less than ten copies of a given protein at steady state).

In order to establish a latent infection, a virus must be a DNA virus. Not all DNA viruses can establish latent infections, but those that do do so as DNA. There is one exception that proves the rule, the retroviruses, which are RNA viruses that then transcribe their RNA into DNA when they establish a latent infection.

In some cases, like bacteriophage lambda or HIV, the latent virus actually inserts itself into the host cell's genome.

In other latent infections, the viral DNA remains in the nucleus, quiet, but not integrated into the host genome. Herpesviruses behave in this manner. The virus may stay in latency for many years (the varicella-zoster virus, a herpesvirus, can stay latent for decades before reactivating to cause zoster [shingles]).

I will point out that latent infection is not the same as persistent infection. Hepatitis B and C can cause persistent infections, but they are not latent. They are constantly infecting liver cells and replicating in them and so the immune system is constantly fighting that process, which leads to chronic inflammation of the liver (chronic hepatitis), which ultimately tends to lead to liver cancer.

Replication

I was a bit scared of writing this section because there are so many different kinds of viruses and they all have so many different fashions in which they replicate, so I'm just going to cover some basics.

Before we begin, I need to review some basics of molecular genetics.

*One gene codes for one protein (that's not actually strictly true, but it'll do for the purposes of this discussion). Generally, the gene and the protein have the same name. So the gene ABC1 codes for the protein Abc1.

*A gene consists of an "Open Reading Frame" (ORF) that starts with a START codon (AUG) and ends with a STOP codon. Before the gene, in a region called the 3' ("three-prime") untranslated region or the 3'-UTR, there will be some sequences that regulate the expression of the gene.

*That bit about one gene coding for one protein? In viruses, it can be true, but often one gene codes for a single "polyprotein" that will then be cut into individual pieces by a viral enzyme called a protease. That's why so many antiviral drugs, like those against HIV, are called protease inhibitors.

OK, now that we've established those basics, proteins in a virus can be classified as "structural" and "non-structural." As a general rule, structural proteins are found in the virus particles, while non-structural proteins are involved with the replication of the virus inside the cell.

Genes in the virus are usually classified as "early" or "late." Early genes are expressed soon after the virus enters the cell and usually are nonstructural proteins involved in copying the virus's genetic material and processing proteins made from those genes, while late genes are usually structural proteins and genes involved in allowing the virus to exit the cell.

Early genes might include genes that replicate the viral genetic material. Often, the first early genes are only one or two large ORFs that make a large "polyprotein." One end of the polyprotein might be a protease that then curls back on the polyprotein and chops it into bits. Other early genes might inhibit host gene transcription, or block certain host factors that might usually serve to recruit an immune response.

Late genes are usually the structural elements of the virus particles themselves. Depending on the virus, they might assemble on little vesicles within the cell. Sometimes aggregates form in the cell while other components assemble on the cell membrane and then these aggregates are packaged into little particles that bud off the membrane. For some viruses, late in the replication cycle you can see under the microscope large "inclusion bodies" which are collections of thousands upon thousands of virus particles waiting to be released.

Egress

Egress can occur in many ways. Some viruses, like adenovirus and bacteriophage T4 literally break the cell open and release intact virus particles. This process is called cell lysis.

For other viruses, like HIV (movie in link 1) the interior components of the virus (the endocapsid) assemble near the membrane and then the virus particle buds off. Ebolavirus forms a really long bud.

Influenza and coronaviruses form inside of vesicles inside the cell and then are transported to the surface and released (pic 1 for influenza and 2 for a feline coronavirus).

And that is the viral replication cycle.

(1)http://scienceofhiv.org/wp/?portfolio=hiv-egress
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Re: A series of posts about virology

Thu Apr 02, 2020 5:39 pm

DocLightning wrote:
For Norovirus, a virus that causes vomiting and diarrhea and is infamous for breaking out on cruiseships, it encounters the host through feco-oral transmission (a microscopic particle of poop on your food).


Message received. I am not getting on any cruise ships.
 
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Re: A series of posts about virology

Thu Apr 02, 2020 5:43 pm

Detecting Viruses

DETECTING VIRUSES.

In Biology, there is a concept called an "assay." An assay is a way of detecting something. A good part of the design of any biological experiment is setting up the assay that will answer your scientific question. So we'll be coming back to that word a lot.

OK, so let's talk about different ways of detecting viruses. But before we begin, it is important to understand that not every virus particle is infectious. For an alphavirus like Semliki Forest Virus, almost every particle is infectious. But for a papillomavirus, only one in about ten thousand particles are infectious. There are a number of reasons for this, but for now, just know that fact.

1) MICROSCOPY
Most viruses (other than poxviruses and a few others) are much smaller than any wavelength of visible light, so they cannot be seen under light microscopy. But electron microscopy can be used as a way to identify the presence of viruses. Here is an electron micrograph of adenovirus particles.

Image

Electron microscopy is time-consuming and expensive, and it takes a very expensive electron microscope (most hospitals don't have one) and specially-trained personnel, so it isn't usually used in clinical settings. However, the first identification of SARS-CoV-2 in Wuhan came from taking samples from patients' lungs and examining them under an electron microscope. The doctors there found coronavirus particles in there and realized that they had a new coronavirus on their hands.

Electron microscopy is also useful in determining what percent of virus particles are infectious because it is possible to directly count the virus particles in a given sample.

2) PLAQUE-FORMING ASSAY

Consider a continuous "lawn" of cells covering the surface of a culture place. These cells might be a bacterium like E. coli growing on agar or they might be a tissue culture cell line like Vero cells or HeLa cells.

If you were to drop a single infectious virus particle on top of that lawn of cells and leave it for some time (usually a day or two) you would come back and see that a "plaque" has formed where that infectious particle fell. This is because the virus particle infected a cell, which then made a bunch more virus particles that infected all the cells around it, and so-on. If you let this go long enough, the plaque would theoretically consume the entire plate. Now, remember that not all virus particles are infectious, so virologists use a term to describe a particle that is infectious: a "plaque-forming unit," or a PFU. (For bacteria, microbiologists use a similar concept: a colony-forming unit or CFU).

So now, we can imagine that there is a ratio implied in this little fact. If not all virus particles are infectious, but an infectious particle is a PFU, then the ratio is the "particle-to-PFU" ratio.

So suppose you have a sample that contains virus and you want to know how many PFUs there are in that sample. Well, you get out a set of culture plates with a lawn of cells and then you drop some of the sample on it. Then you dilute the sample x10 and do that again, and dilute it x10 and do it again... On the first few plates with undiluted or minimally diluted sample, you will probably have so many plaques that the entire plate will die. But as you go down serial dilutions, you will get to a point where there few enough plaques to count. So if you started with a 1mL sample and you got down to 8 plaques on the 7th dilution x10 then you have 8x10^7 PFU/mL.

The trouble with this test is that it is 1) expensive (tissue culture is not cheap) 2) labor intensive and 3) time-consuming (once everything is set up, it usually takes 1-3 days to get a result). But it is useful in certain clinical settings and quite useful in research and also, it is capable of detecting actual infectious virus particles, which many of the other assays cannot do.

Here's a picture of a plaque-forming assay.

Image

3) ANTIBODY ASSAYS

Because viruses have proteins on their surfaces, it is possible to raise antibodies against them, even if they are viruses that do not infect animals. If you inject a mouse with enough bacteriophage T4, which cannot infect a mammalian cell, the mouse will make antibodies against bacteriophage T4. You can then find the gene for one of those antibodies and express it in an expression system and use those antibodies to detect the presence of the virus.

I'm not going to go into the details of how immunoassays work, but basically, an antibody against the viral protein is attached to some sort of label. Perhaps the label is an enzyme that changes the color of a chemical. Perhaps the label is radioactive (used in research). Perhaps the label is a particle of silver or gold that will make a visible mark on a test strip.

Rapid flu and RSV tests work on an antibody assay. There's a technique called ELISA that can be used to detect how many virus particles are present. But these tests cannot actually detect how many of the virus particles are infectious.

The other kind of antibody assay doesn't detect the virus directly but rather detects whether the patient has antibodies against the virus. The basic HIV test checks for antibodies against HIV. The only people who have those antibodies (with a few very rare exceptions) are people who have been exposed to HIV. Some of the newer coronavirus tests will also work this way.

4) AGGLUTINATION ASSAY

Some viruses, most famously influenza, have a protein called hemaglutinin. This protein binds to a receptor on the surface of cells called sialic acid. While no virus can infect a red blood cell (red blood cells don't have ribosomes to make new virus proteins), if influenza virus is added to a sample of red blood cells, it tends to make them clump together and stick to the sides of the test tube. This technique is pretty messy and time-consuming and has been largely supplanted by antibody assays.

5) PCR

PCR stands for the Polymerase Chain Reaction. Reference (1) is the Wikipedia article for the basics of this technique so that I don't have to explain the whole thing.

In order to perform a PCR assay, you first need to know at least some of the sequence of the virus's DNA or RNA. You can then design "primers" (short bits of DNA) that bind specifically to DNA or RNA sequences that are found in the virus of interest and *only* in the virus of interest. In the case of an RNA Virus, the first cycle has to be done using an enzyme from HIV called reverse transcriptase to transcribe the viral RNA into DNA and after that, you can continue with regular PCR. This technique is called RT-PCR. Using PCR, you can then start to make copies of the sequence between the primers, and then copies of the copies, and then copies of the copies of the copies until you have billions or even trillions of copies.

Initially, PCR was a "qualitative" test and it was quite time-consuming (took a couple of days to run the cycles of PCR and then the sample had to be run out on an agarose gel). If a piece of DNA of the appropriate size appeared, then the virus was determined to be present.

But now, the test can be done in a different way. A dye is introduced into the reaction tube (I've attached a picture of a PCR tube). This dye is fluorescent and it only fluoresces when it binds to double-stranded DNA. So after each round of PCR, a specific frequency of light is shone into the tube and a detector looks for a specific frequency of fluorescence. The fewer number of cycles it takes to detect the fluorescence, the more viral DNA or RNA was in the sample to start.

Image

There is a big pitfall with PCR-based tests: the test looks for the viral genetic material, but NOT for actual infectious virus. Not only that but with the SARS-CoV-2 test, the test looks for two or three specific sequences in the viral genome and if it finds any of them, the test is considered positive. So that means that you can have a patient who had COVID-19 and has completely recovered, and yet still tests positive over a month later because little fragments of viral RNA are still being coughed up. This is thought to be the explanation for some patients who tested negative and then tested positive again or for patients who were testing positive even over a month after recovering.

(1)https://en.wikipedia.org/wiki/Polymerase_chain_reaction
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Re: A series of posts about virology

Thu Apr 02, 2020 5:50 pm

Thanks for this series of posts, by the way. Very informative and readable.
 
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Re: A series of posts about virology

Thu Apr 02, 2020 6:20 pm

How (or why) does the sex pilus, just a series of proteins, "break off" it's donor cell?

Why would it be safe to argue that plasmids, "though just loops of DNA", would not be alive? Isn't genetic material "alive"?

Sorry for the 9th grade questions.
 
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Re: A series of posts about virology

Thu Apr 02, 2020 7:23 pm

N212R wrote:
How (or why) does the sex pilus, just a series of proteins, "break off" it's donor cell?

Why would it be safe to argue that plasmids, "though just loops of DNA", would not be alive? Isn't genetic material "alive"?


The sex pilus does not break off the donor cell. That was more of a theoretical exercise.

As for why a plasmid isn't alive, not all that reproduces is alive and not all that carries information is alive. A computer virus satisfies both criteria and it's not alive.

Life is an emergent property of a number of non-living components working together. DNA is just a molecule and without a living system surrounding it, it cannot do much of anything.

There are places where the line gets blurred. The genus of bacteria Chlamydia are obligate intracellular parasites. They do not have the ability to generate their own energy and so they must be surrounded by a living cell (in nature) in order to reproduce. That said, they *can* be grown without a living system if they are provided with enough ATP.

Poxviruses are a lot like a degenerate cell. They have their own DNA-dependent RNA polymerase, but new virus particles are still assembled from components, rather than budding or dividing like a bacterium.

But as a rule, life has to come from life. So when a cell divides, a collection of *ALL* the necessary components must be assorted into the daughter cell, not just DNA. And that's not the case for a plasmid.
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Re: A series of posts about virology

Thu Apr 02, 2020 7:53 pm

DocLightning wrote:
There is a big pitfall with PCR-based tests: the test looks for the viral genetic material, but NOT for actual infectious virus. Not only that but with the SARS-CoV-2 test, the test looks for two or three specific sequences in the viral genome and if it finds any of them, the test is considered positive. So that means that you can have a patient who had COVID-19 and has completely recovered, and yet still tests positive over a month later because little fragments of viral RNA are still being coughed up. This is thought to be the explanation for some patients who tested negative and then tested positive again or for patients who were testing positive even over a month after recovering.


So in converse, because they only pick a few chains, if a company rushes a test out for the virus, they could be looking at chains that occur in other viruses or naturally causing false positives. Correct?
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Re: A series of posts about virology

Thu Apr 02, 2020 8:55 pm

Were virologists, on the lookout for the next big outbreak, predicting a virus with a shared receptor to the original SARS or would that have been unexpected?

What did MERS use as a receptor?



.
 
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Re: A series of posts about virology

Thu Apr 02, 2020 9:30 pm

casinterest wrote:
So in converse, because they only pick a few chains, if a company rushes a test out for the virus, they could be looking at chains that occur in other viruses or naturally causing false positives. Correct?


Unlikely. They choose primers that are directed against truly unique sequences in this virus. Now, could there be some other virus out there that we don't know about with this sequence? Sure. But given the fact that there's a pandemic right now, the chances of accidentally detecting one of those are rare and also, the diagnosis shouldn't be based *only* on the test.

N212R wrote:
Were virologists, on the lookout for the next big outbreak, predicting a virus with a shared receptor to the original SARS or would that have been unexpected?

What did MERS use as a receptor?


So indeed a paper in 2007 warned of just this kind of thing(1) and they predicted that the ACE2 receptor was a likely target. But nobody listens to scientists...

Interestingly, MERS-CoV uses DPP4, a different receptor and it still occurs. Person-to-person transmission is rare but camel-to-person transmission is the usual fashion in which human infections occur.

(1)https://cmr.asm.org/content/20/4/660
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Re: A series of posts about virology

Thu Apr 02, 2020 10:53 pm

Are you going to do a follow up post about the race for the vaccine? I am interested a bit more in the functionalities involved there. IE what constitutes a "dead" virus? Do they find a way to block the "spike" proteins?
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Re: A series of posts about virology

Fri Apr 03, 2020 12:44 am

On the Structure and Function of Virions

A virion, or virus particle, is a vehicle that transports the viral genetic material from one cell to the next or one host to the next. The structure of the virion varies from virus to virus and his structure determines the means by which viruses are transmitted. That said, there are some general themes that are common to most viruses.

So, from the inside out:

1) Genetic Material

In the core of the virus is the genetic material. For one very strange kind of virus found only in plants (a viroid) that's all there is. No coat or anything. But for most viruses, this genetic material is a) associated with a set of "nucleoproteins" that help to package, organize, and protect the genetic material (RNA in particular tends to be pretty fragile stuff) and b) surrounded by another layer of...something.

2) Endocapsid

Many viruses have a protein shell that is not the outermost layer of the virus. This is called the endocapsid or nucleocapsid. In the diagram of influenza virus shown here, the endocapsid is labled "capsid." This layer can serve any number of functions. It can serve as a rigid skeleton for the virion out of which surface proteins are coordinated. It can play a key role in the initial stages of cell entry after the virus particle has made its first step into the cell. It can also coordinate certain proteins that are necessary for the first steps of viral replication in the cell.

https://micro.magnet.fsu.edu/cells/viruses/images/influenzafigure1.jpg

In other viruses like norovirus this structure is the outermost layer of the particle, and so rather than being called an "endocapsid," it is simply the capsid.

Image

For many viruses, these capsids take on an icosahedral shape. An icosahedron is a 20-sided solid, the largest platonic solid that can be formed of regular sides (tetrahedron, cube, octahdron, dodecahedron, icosahedron). This is important because viruses usually contain only a relatively small amount of information, so there is selective pressure to be able to build the largest physical structure out of the smallest amount of information.

And yet not all viruses follow that rule. The filoviruses (which include ebola) arrange their nucleocapsid into a helical shape, which gives them their characteristic filamentous shape.

Image

3) Envelope

Not all viruses are enveloped. However, for those that are, the envelope is made of phospholipid bilayer membrane, which I covered in an earlier post.

In some enveloped viruses, like influenza, proteins that stick through the envelope are coordinated by endocapsid proteins.

An advantage of being enveloped is that it is a simpler matter for the viral envelope to fuse with the cell membrane to initiate the infectious cycle. But a disadvantage of being enveloped is that the virus particle is more fragile.

For example, norovirus (unenveloped) can remain stable on surfaces for days, even weeks and is resistant to being dried. It is stable against detergents and alcohol. Only bleach, heating above 60C, and incineration can destroy the virus particle. By contrast coronaviruses (enveloped) disappear when exposed to detergents or alcohol.

Sructure of a Coronavirus Virion

Coronaviruses are a bit unusual in their structure because they do not have a classical icosahedral endocapsid.

1) Genetic Material

Coronaviruses have one of the largest genomes of any known RNA virus at about 30kb (kilobases, not kilobytes). Rather than having an icosahedral nucleocapsid, a coronavirus contains a nucleocapsid protein (N) that binds to and stabilizes the RNA (left to its own devices, a molecule of RNA tends to ball up into a tangled mess of various intermolecular interactions). These nucleocapsid-RNA complexes are then bunched up against a membrane in which there are three proteins, E, M, and S. As a result, coronaviruses are "pleiomorphic" (different shapes) with some particles a bit bigger or smaller than others because there is no rigid icosahedral geometry to form a traditional nucleocapsid.

2) Structural Proteins

E is the envelope protein. It is not entirely clear what its function is, but coronaviruses that are genetically engineered to lack this protein can still infect cells, albeit not as well. It might serve as an ion channel that gets used during the intracellular replication process and it seems to facilitate assembly and release of virus particles. In fact, a SARS immunization was designed with a genetically-engineered virus deficient in E and this protected animals from severe respiratory disease on challenge with SARS-CoV(1)

M is the membrane protein. It seems to lend curvature to the membrane and it may be involved in binding the N protein to the inside of the membrane.

The S protein is perhaps the most famous. S stands for "spike" and these proteins are a series of club-like projections that stick out of the envelope. The spike protein serves two functions: a) it tethers the virus particle to the cell by interaction with a receptor (for SARS-CoV-2 that receptor is the ACE2 protein) and b) it contains in its interior a set of structures that cause the envelope of the virus to fuse with the target cell.

Finally, some coronaviruses contain hemagglutinin esterase (HE), which is thought to enhance binding of the virus particle to the host cell. SARS-CoV-2 does not have this protein.

Image

(1)https://www.ncbi.nlm.nih.gov/pubmed/20110095/
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Re: A series of posts about virology

Fri Apr 03, 2020 1:09 am

I think a few are wondering silently about the question I put forward here now...

Have our (as in man, not Americans/Russians/Chinese) medical technological innovation risen to the point it is possible to create a synthetic virus or like agent to act as one?

This question comes from the fact that plentiful men around the globe without morals, principles, ethics, compassion for other human beings.

We know many former USSR individuals, now oligarchs, hold tons of weaponry seized, sold and stolen after the USSR fall...

..if they had a Ft. Detrick type installation somewhere or multiple locations to develop bio-tech weapons. So to the best of your knowledge, given these labs have been around for decades, can a man made virus be developed?

Feel free not to answer this given your position and who knows who is scanning this site for 'talkers'...

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Re: A series of posts about virology

Fri Apr 03, 2020 1:33 am

DocLightning wrote:
Because viruses have proteins on their surfaces, it is possible to raise antibodies against them, even if they are viruses that do not infect animals.


My understanding is the horseshoe bat can be infected with the SARS virus but doesn't get sick? What do we know about that mechanism?
 
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Re: A series of posts about virology

Fri Apr 03, 2020 2:18 am

Can you please explain "gain of function mutation" and why it is, I believe, a very controversial subject?

Thanks much for your excellent overview.
 
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Re: A series of posts about virology

Fri Apr 03, 2020 2:31 am

N212R wrote:
Can you please explain "gain of function mutation" and why it is, I believe, a very controversial subject?

Thanks much for your excellent overview.


A gain-of-function mutation is a mutation that makes a certain protein work "better" than it is supposed to. That isn't always a good thing because most biological systems are based on tight regulation. As an analogy, imagine a malfunction in a FADEC system that made the engine run at 100% N1 all the time. "This engine works BETTER than a normal engine!" Well, that's not such a good thing.

I have a patient with a gain-of-function mutation in a sodium channel in his central nervous system. Sodium channels make neurons fire. Well, the result of his little mutation is intractable seizures. The neurons fire *way* too much and his brain has basically wasted itself away until the seizures stopped. Or imagine a gain-of-function in the receptor for insulin that made the receptor behave as if it had bound insulin all the time. That would probably be a mutation that is incompatible with life.

And I am not aware of a controversy about gain-of-function mutations. Care to fill me in?
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Re: A series of posts about virology

Fri Apr 03, 2020 2:44 am

BN747 wrote:
I think a few are wondering silently about the question I put forward here now...

Have our (as in man, not Americans/Russians/Chinese) medical technological innovation risen to the point it is possible to create a synthetic virus or like agent to act as one?


As far as I know, no. We *still* don't understand exactly what molecular features of this virus have made it so successful. There is a lot of complexity between the genetic code of the virus and its clinical behavior and presentation.

So from the point of a phenotype (the observed characteristics), we know that this virus is successful because it is deadly in only a small percentage of patients (but enough to wreak havoc) and because there is a large portion of patients who are infected and have minimal to no symptoms, some of which are because they are in the incubation period and some of which are because they just don't get that sick. As compared to SARS, which made people extremely sick within 48 hours usually, this virus spreads much more easily.

In fact, the most successful viruses don't even make us sick. Circoviruses (you've probably never heard of them) infect almost all of us and yet they cause no symptoms, so they silently have spread all over the world and nobody cares to stop them. Certainly, there is a strong selective pressure on most viruses to cause mild symptoms because a virus that kills its host will not spread well. That's why Ebola never really became a major pandemic.

But we still do not understand the precise genotypic features that grant SARS-CoV-2 this behavior. This is an area of active research because we would like to know if there is a way to predict which viruses have the potential to cause pandemics.

From the point of a bioweapon, this one would be a poor choice. It has reached every continent except Antarctica (and I would be willing to bet it will reach there, too). And yet, it is not a doomsday weapon, for the world will return to normal after this has passed (and it will pass). It has destroyed the military readiness of every military in the world. For a bioweapon, you'd want one that will make a lot of people sick really quickly and so that way it stays put.

The virus that has me most worried is Marburg, which is similar to Ebola (a filovirus with similar genetics and clinical presentation) and has a lethality of about 88%. I believe Russia has a stockpile. The good news is that we can make a vaccine against Ebola, so in a pinch, we could have one against Marburg probably in a couple of months just by using the same techniques. If I were Supreme Dictator for Life, I'd have someone working on that vaccine now.

But to have come up with a virus such as this...there is too much complexity for even the most powerful computers to predict this behavior.
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Re: A series of posts about virology

Fri Apr 03, 2020 2:57 am

DocLightning wrote:

But to have come up with a virus such as this...there is too much complexity for even the most powerful computers to predict this behavior.


Thanks for that insight.

As far as contemporary computers go true, a quantum computer is a whole other ball game. Supposedly still being developed, executes unlimited computations in mere seconds, the 1st wind I got of this was like 10 years ago.

A MIT professor was spearheading that push then..some progress surely has been made in the last 10 years.

One lesson I learned from military life is this, we some new tech is 'released'..in truth, it's been under hush hush for at least 10 to 15 years. I have a decent chunk of years left on this rock (car accidents withstanding) and I do believe quantum computers will be online before I go, but in consumer models..not a chance!

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Re: A series of posts about virology

Fri Apr 03, 2020 3:10 am

BN747 wrote:
As far as contemporary computers go true, a quantum computer is a whole other ball game. Supposedly still being developed, executes unlimited computations in mere seconds, the 1st wind I got of this was like 10 years ago.


As far as quantum computers go, they won't be of much help unless we know what to model in the first place...and we do not understand the entire human body to that level of granularity.
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Re: A series of posts about virology

Fri Apr 03, 2020 3:24 am

DocLightning wrote:
BN747 wrote:
As far as contemporary computers go true, a quantum computer is a whole other ball game. Supposedly still being developed, executes unlimited computations in mere seconds, the 1st wind I got of this was like 10 years ago.


As far as quantum computers go, they won't be of much help unless we know what to model in the first place...and we do not understand the entire human body to that level of granularity.


I think is the logjam being sorted out as we speak. Of course since it's of human design we are sure to overlook some key necessary security sectors.

In the mid 1980s, I received for my office the most advanced CRS systems available.

Number one waste of time on it? Looking at who was flying out every AA flight nonstop LAX to JFK. Why?

We were able to see who was in FC. Michael Jackson. His assigned seat, address of record, phone contacts and credit card payment info.
That info was available for every passenger on flights. Looking back, were I of a criminal mind..I could have caused a lot of damage (and profitable) and heartache...but I was astonished at what was at my finger tips in the early days. We will make the same error-prone oversight mistakes with quantum computers when we get there. We're humans...'nuff said.

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Re: A series of posts about virology

Fri Apr 03, 2020 4:20 am

DocLightning wrote:
And I am not aware of a controversy about gain-of-function mutations. Care to fill me in?


It would appear that not everyone is happy with "gain of function" research.

https://www.change.org/p/national-insti ... _dashboard

Moreover, the NIH (National Institute of Health) has been less than laudatory about the practice.

http://www.cidrap.umn.edu/news-perspect ... r-guidance
 
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Re: A series of posts about virology

Fri Apr 03, 2020 4:01 pm

DocLightning wrote:
E is the envelope protein. It is not entirely clear what its function is, but coronaviruses that are genetically engineered to lack this protein can still infect cells, albeit not as well. It might serve as an ion channel that gets used during the intracellular replication process and it seems to facilitate assembly and release of virus particles. In fact, a SARS immunization was designed with a genetically-engineered virus deficient in E and this protected animals from severe respiratory disease on challenge with SARS-CoV(1)


Seems that genetic engineering has been a well trod path by which virologists have attempted to unlock some of the mysteries of these coronaviruses and most especially the SARS coronavirus. Would that be a fair statement?

In fact, the researchers in Wuhan were at the "cutting edge" of such manipulations. What are the chances that the hitherto unknown "variant' virus SARS-CoV 2 reared it's ugly head directly in their backyard? Talk about a blessing AND a curse...

Full "conspiracy" disclosure; I don't believe the deadly viral genetic material was transmitted to humans via an animal host.

Would like to read your well-educated supposition as to this still unresolved question.
 
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Re: A series of posts about virology

Fri Apr 03, 2020 4:06 pm

N212R wrote:
In fact, the researchers in Wuhan were at the "cutting edge" of such manipulations. What are the chances that the hitherto unknown "variant' virus SARS-CoV 2 reared it's ugly head directly in their backyard? Talk about a blessing AND a curse.


Through what peer-reviewed analysis have you arrived at this purported ‘fact’? Inquiring minds would like to know.
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Re: A series of posts about virology

Fri Apr 03, 2020 5:53 pm

N212R wrote:
Seems that genetic engineering has been a well trod path by which virologists have attempted to unlock some of the mysteries of these coronaviruses and most especially the SARS coronavirus. Would that be a fair statement?

In fact, the researchers in Wuhan were at the "cutting edge" of such manipulations. What are the chances that the hitherto unknown "variant' virus SARS-CoV 2 reared it's ugly head directly in their backyard? Talk about a blessing AND a curse...

Full "conspiracy" disclosure; I don't believe the deadly viral genetic material was transmitted to humans via an animal host.


Well...all the data to date say that you're wrong. There are known methods of doing genetic engineering and those methods leave their marks. Those marks are absent here.

The high degree of sequence homology to existing bat coronaviruses and to the prior SARS coronavirus suggest a natural origin.

The paper that goes through this reasoning is here: https://www.nature.com/articles/s41591-020-0820-9

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Re: A series of posts about virology

Fri Apr 03, 2020 6:18 pm

DocLightning wrote:
There are known methods of doing genetic engineering and those methods leave their marks. Those marks are absent here.


Would you care to expound?

The high degree of sequence homology to existing bat coronaviruses and to the prior SARS coronavirus suggest a natural origin.


Let us know when suggestion can be scientifically homologized.

The paper that goes through this reasoning is here: https://www.nature.com/articles/s41591-020-0820-9

You don't have to believe it...but science doesn't care what you believe.


ONE article from the folks better known for fancy magazine covers than rigorous scientific method? Please tell us about the hallmark scientific precedents first published in such a generalist journal?
 
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Re: A series of posts about virology

Fri Apr 03, 2020 6:23 pm

N212R wrote:
ONE article from the folks better known for fancy magazine covers than rigorous scientific method? Please tell us about the hallmark scientific precedents first published in such a generalist journal?

Love your chutzpah Mr. One Article:
N212R wrote:
https://www.zerohedge.com/health/one-worst-coverups-human-history-msm-turns-gaze-chinese-biolab-near-covid-19-ground-zero

Time to spool up the engines, get up to speed...and question much received "wisdom".


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Re: A series of posts about virology

Fri Apr 03, 2020 9:07 pm

N212R wrote:
ONE article from the folks better known for fancy magazine covers than rigorous scientific method? Please tell us about the hallmark scientific precedents first published in such a generalist journal?


And your background in molecular virology that gives you the training to evaluate this article is...?

Believe what you want. I will not answer more of your questions.
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Re: A series of posts about virology

Fri Apr 03, 2020 11:17 pm

Extremely informative and interesting. Thank you DocLightning!
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Re: A series of posts about virology

Fri Apr 03, 2020 11:23 pm

N212R wrote:
DocLightning wrote:
There are known methods of doing genetic engineering and those methods leave their marks. Those marks are absent here.


Would you care to expound?

The high degree of sequence homology to existing bat coronaviruses and to the prior SARS coronavirus suggest a natural origin.


Let us know when suggestion can be scientifically homologized.

The paper that goes through this reasoning is here: https://www.nature.com/articles/s41591-020-0820-9

You don't have to believe it...but science doesn't care what you believe.


ONE article from the folks better known for fancy magazine covers than rigorous scientific method? Please tell us about the hallmark scientific precedents first published in such a generalist journal?


Let us know when you have a peer-reviewed source for your fishing expedition.
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Re: A series of posts about virology

Sat Apr 04, 2020 3:34 am

Found an interesting site regarding diagnostics, treatments, and vaccines:

https://www.av.co/covid

Image

Image
 
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Re: A series of posts about virology

Sat Apr 04, 2020 8:24 am

Doc Lighting,

First off thank you for taking the time to post. You're masterfully at teaching.
my curiosity has been aroused about this virus that's been wreaking havoc worldwide.
It's nice to hear from someone who's grounded and has a medical background and no
objective, other than to teach.
 
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Re: A series of posts about virology

Sat Apr 04, 2020 8:31 pm

I have a decent background in biophysics and statistics, some publications in those fields. I work on advertising analysis and customer analysis, think Facebook, Google, etc. I am glad to see this virology for when I have any brain cells left to try to re-learn cell biology and organic chemistry. Most likely it is way too late for me until I retire. What this thread inspires me to do is to do a series on experimental design, that could be used for epidemiology, which is a close sister to what my coworkers do. Facebook would also have unbelievably skilled epidemiologists working there.

Long story short, you can't test people around a hospital and get a "virus count," then test dead people and get a "death count." That is not how experimental design, epidemiology or medical pathology works. That's not how any of it works. Okay, not to hijack your thread. I can't absorb virology, but those are some elegant pictures and diagrams. We used to have a tool called Rasmol to do those.
 
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Re: A series of posts about virology

Sat Apr 04, 2020 11:27 pm

N212R wrote:
DocLightning wrote:
And I am not aware of a controversy about gain-of-function mutations. Care to fill me in?


It would appear that not everyone is happy with "gain of function" research.

https://www.change.org/p/national-insti ... _dashboard

Moreover, the NIH (National Institute of Health) has been less than laudatory about the practice.

http://www.cidrap.umn.edu/news-perspect ... r-guidance


Oh, THAT. Mixed bag. I think that we can glean useful information from such research. I also think that this information can be badly misused and of course, there can be an...accident.

But generally, I tend to frown on those who want to stop scientific research, and not just because I'm a former scientist and I think that all science is good. *IF* that information exists, then it can and will be discovered. Now, do we want that to happen in a tightly-regulated and monitored setting in the USA, or do we want that to happen in some half-assed lab in some nation with less rigorous ethical and technical standards?
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Re: A series of posts about virology

Sat Apr 04, 2020 11:44 pm

A Brief History of Viruses

Origins

Viruses are too small to leave fossils and because many of them are RNA viruses and RNA is unstable stuff, their original genetic sequences are not preserved in the fossil strata.

We can make some extrapolations about the origin and history of viruses by comparing their modern sequences and drawing tress of phylogeny, but the origin of viruses will remain a question that cannot be answered.

Moreover, there are are several different hypotheses about the origins of viruses and it's likely that more than one of them is true, since it's likely that viruses arose through a number of different mechanisms.

1) Prebiotic/RNA World

RNA is unstable and a poor medium for the long-term storage of genetic information. But it is also a versatile molecule. RNA can form complex, three-dimensional structures and can function as an enzyme. This is called a ribozyme. The ribosome, that cellular organelle on which proteins are generated is an RNA structure and while it has proteins in it, there are many ribozyme functions. Not only that, but RNA polymerase ribozymes that can replicate RNA have also been described.

Image
A ribosome, showing a ribozymatic catalytic site in detail

One hypothesis is that the first self-replicating molecules on earth were RNA molecules that formed ribozymes and managed to replicate themselves (or each other).

Image
The catalytic core of a ribozyme that can produce new RNA (an RNA polymerase ribozyme).

In time, lipid membranes formed and various proteins became involved, a massive jump from RNA to DNA occurred, and the first cellular life forms emerged. According to this hypothesis, many RNA viruses are the descendants of this prebiotic RNA world.

2) Degeneracy

The bacteria rickettsia and chlamydia are obligate intracellular parasites. The chlamydiae were once thought to be viruses, but they have their own membranes and ribosomes and all of the proteins and enzymes they need to encode and utilize their own genetic material, a property not found in any virus. What they lack is the machinery to produce their own ATP, that famous molecule that "powers the cell." Rather, they rely on ATP from the host cell to drive their own internal mechanisms.

Poxviruses are complex and large DNA viruses. They contain their own DNA, their own DNA-dependent RNA polymerase, their own DNA-dependent DNA polymerase, and many other enzymes that most viruses lack. During intracellular replication, a poxvirus sets up a "counter-nucleus" to the cell nucleus that directs the cell to generate more poxviruses. However, unlike the rickettsiae and chlamydiae, poxvirus particles are assembled within the cell, rather than being produced by budding or binary fission.

Genetic evidence suggests an evolutionary to adenoviruses or perhaps an ancient and now extinct poxvirus, but could poxviruses be degenerate cells?

3) Escape/"Vagrancy"

In one of my first virology posts, I discussed plasmid F and its sex pilus. I raised the question of what would happen if some sequences mutations in plasmid F both a) extended the pilus to be long enough to carry the entire plasmid and b) allowed the pilus to detach from the donor cell so that it could float freely.

This would be an example of "escape" of a cellular set of genes to become a virus. There are problems with this hypothesis, because some viruses contain genes that are not analogous or homologous to any cellular genes.

4) Coevolution/"Bubble" Hypothesis

This hypothesis does not contradict any of the first three, but supposes that viruses around the same time as cellular life. According to this hypothesis, the first replicating molecules, called replicons, would have formed in areas rich in resources (near hydrothermal vents, for example). Further away from these resource-rich environments, replicons became encased in "bubbles" of biologic membranes that helped to concentrate the resources that they needed. Some of these replicons stayed within their membranes, while others would enter these membranes, utilize their resources, and then depart to another membrane. This second group would have eventually gone on to become viruses.

5) Chimeric Origins

This hypothesis is a combination of the "Escape" and "Prebiotic" hypothesis in which proto-viruses might have moved from host to host and acquired their structural and nonstructural proteins in that manner.

History:

A carving from ancient Egypt depicts a man with a withered leg, likely from polio. An engraving from the time of the Spanish conquest of Mexico shows a victim of smallpox. Chickenpox and roseola, both diagnoses instantly recognized by generation upon generation of grandmothers. An image just over a century old of sick and dying lying in their beds in a makeshift hospital, struck by an illness that had a name but no identified cause. Microscopic examination of the blood of the victims frequently showed the presence of a gram-negative bacterium, which those physicians named "Haemophilus influenzae." They did not know that this bacterium was merely a passenger that had taken advantage of a sickened and weakened body.

Image
An engraving from ancient Egypt showing a man with a withered leg, perhaps from polio

Image
An engraving from Mexico around the time of the Spanish conquest showing native victims of smallpox

Image
A photograph showing a field hospital during the 1918 influenza pandemic

Viral diseases have been described through human history and prehistory. Louis Pasteur searched in vain for the cause of rabies but prophetically concluded that perhaps it was an agent too small to be seen by microscopy. In 1886 Dr. J. Buist of Edinburgh was the first person to see virus particles in vaccinia vaccine lymph, particles he described as "micrococci" and mistook for bacteria. In 1892, the Russian botanist Dmitry Ivanovski found that an extract from the leaf of a tobacco plant affected by tobacco mosaic disease could be passed through a Chamberland filter-candle, which was capable of filtering out bacteria, and yet this filtrate remained able to infect new tobacco plants with the disease. But Ivanovski did not grasp the full meaning of this discovery. Six years later, in 1898, Martinus Beijerinck, a Dutch microbiologist found that this incitant could diffuse through an agar gel, an "contagium vivum fluidum," something that was dissolved in water, unlike an "contagium vivum fixum," a bacterium. He thought that perhaps this contagium was a fluid, but he coined the term "virus." That same year, the German biologists Friedrich Loeffler and Paul Frosch passed the first animal virus through the same filter and discovered that the cause of hoof-and-mouth disease (an enterovirus) was also a virus.

By 1928, enough of these "filterable diseases" had been discovered that Thomas Milton Rivers was able to publish a catalog of essays covering all known viruses. Three years later, in 1931, the German engineers Ernst Ruska and Max Knoll invented electron microscopy. This invention changed the science of microbiology forever and is still in use today. For the first time, microbiologists could see bacteriophages in the process of infecting bacteria, and they could see that they were complex structures.

Image
A transmission electron micrograph of tobacco mosaic virus particles

In 1935, Wendell Stanley discovered that the tobacco mosaic virus could be condensed into crystals and he determined that these crystals were mostly made of protein. Five years later, he and Max Lauffer separated these crystals into their components and discovered that in addition to protein, there was RNA. Fred Griffith had discovered bacterial transformation in 1928, but it wasn't until 1944 that Avery, McLeod, and McCarty isolated this "transforming factor" and discovered that it was DNA. It was now apparent that the RNA found in viruses was their genetic material and that other viruses contained DNA as theirs.

With this information, some key information about viruses was deduced. 1) They were particles. 2) They contained nucleic acids as their genetic material. 3) Their protein (and/or lipid) coats were important to their ability to infect cells.

Throughout most of the 20th century, viruses were detected in outbreaks of disease among livestock, agricultural plants, and humans. Samples were taken and examined under electron microscopy and tested with plaque assays. This discovery changed the world. Polio, that ancient scourge, was discovered to be a virus and Jonas Salk created a vaccine. Polio is on the verge of eradication. Vaccines against other viral diseases, like measles, mumps, rubella, varicella, hepatitis A and B, influenza, and rotavirus were developed (Edward Jenner had, of course, invented the first vaccine from the vaccina virus of cowpox, but he did not know what a virus was). One by one, these ancient scourges began to fall.

More recently, since the late 1990s, the advent of advanced molecular genetic techniques has allowed virologists to probe the genetic material of many plants and animals. These new techniques have unveiled an entire world of viruses that live within us and on us. We have a microbiome, also unveiled by these techniques, but we also have a virome. We are only beginning to discover that this virome may have profound implications on human health.

Virology has always been considered an obscure field, one of limited importance to everyday life. And yet today in 2020, the whole world suddenly understands the importance of this small, obscure branch of science. On October 1st 2019, probably less than 1% of the world knew what a coronavirus was. Today, "coronavirus" is a household word.
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Re: A series of posts about virology

Sun Apr 05, 2020 2:43 am

I only remember the word, because of my dog's vaccination records.
 
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Re: A series of posts about virology

Tue Apr 07, 2020 3:43 am

Now with the tigers getting coronavirus, is my dog still OK with his vaccine shot does anybody know?
 
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Re: A series of posts about virology

Thu Apr 09, 2020 3:54 am

Coronaviridae: An Overview

The order Nidovirales contains three families: Coronviridae, Roniviridae, and Arteriviridae. Roniviruses seem to infect shrimp and mosquitos (arthropods). Artiviruses do cause disease in mammals, but none are known to infect humans (although one does cause a hemorrhagic fever in monkeys).
Coronaviruses are a large family of viruses that infect vertebrates. Two genera, the Alphacoronaviruses and Betacoronaviruses, infect almost exclusively mammals, with bats as a major host. Two other genera, the Gammacoronaviruses and Deltacoronaviruses infect almost exclusively birds and a few reptiles with the exception that one Gammacoronavirus infects beluga whales and some Deltacoronaviruses infect pigs.

Genetic sequence analysis of the family Coronaviridae places its origin at about 10,000 years ago. This is difficult to reconcile with the mostly strict taxonomic division between the Alpha- and Betacoronaviruses, which infect only mammals, and the Gamma- and Deltacoronaviruses, which infect mostly birds and a few reptiles. This would suggest that the most recent common ancestor of the coronaviruses originated around 310-320 million years ago when ancestors of mammals (synapsids) diverged from the ancestors of birds and dinosaurs (sauropsids). That said, the fact that there is crossover in viral genera from birds to mammals suggests that perhaps the origin is more recent than 300 million years, but there is still a suggestion of an origin millions, not thousands, of years ago.

In humans, there are seven coronaviruses known to cause clinical disease. Two of these are endemic Alphacoronaviruses with the rather poetic names HCoV-229E and -NL63 and two more are endemic Betacoronaviruses called HCoV-HKU1 and -OC43. These four cause 10-20% of cases of the common cold, and can also cause viral pneumonia in young children or the elderly.

The latter is particularly interesting. Genetic clock analysis puts its emergency around 1890, a time when a widespread pandemic of respiratory illness was infecting cattle and there was mass culling of herds around the world. In 1889-1990 a global pandemic of human respiratory disease was described originating in China. This illness was characterized by fever, cough, malaise, and marked neurologic symptoms. At the time, it was ascribed to influenza, a hypothesis that can never be proven as tissue samples from that time are unavailable. But it is tempting to hypothesize that this pandemic might actually have represented the emergence of a novel zoonotic coronavirus passed from cattle to humans (-OC43 is clearly descended from a bovine betacoronavirus) that attenuated so as to adapt to its new host, which is a common pattern in viral evolution.

The other three human coronaviruses known to cause clinical disease are all Betacoronaviruses. SARS-CoV, related to a bat coronavirus was described in 2003. MERS-CoV was described in 2012, descended from a camelid coronavirus. Finally, today we are faced with a new pandemic strain, SARS-CoV-2, the causative agent of COVID-19, also bat-descended, and we are at over 1.5 million cases and counting.

Virion Structure

I covered the basic structure of the virion in a prior post. To review: coronaviruses are roughly spherical and enveloped by a membrane. They are not icosahedral in arrangement, and so they can vary in shape and size with an average of 80-120nm and extremes of 50-200nm. The spike proteins (S) are club-shaped and emerge from the surface of the virion. These have two functions: they bind to the host membrane receptor and they cause the viral membrane and the host membrane to fuse. Spike proteins in all coronaviruses are “trimers” comprised of three identical components (many viruses use trimeric proteins to bind to host receptors). The S protein consists of two domains, S1 and S2. S1 is the domain that binds to the host receptor and S2 is the fusion peptide. S1 varies widely between coronaviruses, while S2 is very similar across coronaviruses. In some coronaviruses, including SARS-CoV-2, the S1 and S2 peptides are actually split by a protease enzyme while still in the infected host cell.

Two other proteins are found in the envelope. The protein M seems to help to curve the membrane into the appropriate shape and the protein E seems to be important to the successful reproduction of the virus, but it varies greatly from coronavirus to coronavirus and its exact function is unclear.
Finally, in some betacoronaviruses (but not SARS-CoV-2) is a membrane protein called HE for hemagglutinin-esterase. This may help the virus travel through the thick mucus that surrounds many mucus membranes and it may also assist the S protein in its function.

Coronaviruses do not have an icosahedral nucleocapsid. Rather unusually for a positive-sense RNA virus, the nucleocapsid has a helical symmetry. Most helical RNA viruses (Ebola, influenza, RSV) are negative-sense RNA viruses. This nucleocapsid consists of a single protein, N, and the RNA genome.

A schematic is shown here.

Image

Genome Structure and Organization

A gene is a segment of DNA (or RNA for an RNA virus) that codes for a protein. All genes have a few features in common:

1) The coding regions start with a START codon (remember, a codon is a sequence of three nucleotides that codes for a given amino acid).

2) They end with one of three STOP codons.

3) They have a region before the gene called the 5’ (five prime) untranslated region or 5’UTR that helps to regulate expression of the gene and allows the corresponding mRNA to bind to the ribosome.

Any sequence of DNA or RNA that starts with a START codon and ends with a STOP codon is called an “open reading frame” or ORF. Not all ORFs are genes, but all genes have an ORF. It's also important to understand that a "reading frame" is based on reading sets of three-nucleotide codons. If the frame shifts by one or two nucleotides, this will code for a completely different amino acid sequence. These "frame shifts" may be accidental (caused by mutations) or a feature of genes that conserves space by allowing two genes to overlap.

Coronaviruses do not have DNA, nor does DNA ever enter into the replication cycle of a coronavirus. They are remarkable for RNA viruses in that their genomes can exceed 30,000 bases in length (30kb, which stands for “kilobases”). Across the entire family Coronaviridae, the general organization of the genome is conserved (meaning that it varies very little from one to the next). A schematic of some coronavirus genomes is attached.

The first gene at the 5’ end is called ORF1a and ORF1b, or sometimes rep 1a and rep1b. They actually overlap, because RNA sequences can be read differently by shifting the reference frame. This set of genes encode the replicase complex, including the RNA-dependent RNA polymerase that is blocked by the drugs remdesivir (Gilead) and favipiravir (Fujifilm).

After this lie a series of accessory genes, usually numbered (2a, 2b, 3a, 4, etc.), which may include the HE gene, followed by the S, followed by some more accessory genes, followed by the E and M genes (which, curiously, never have any other genes between them), some more accessory genes, and then the N gene at the 3' end. These accessory genes may serve different functions in different coronaviruses depending on the cell type and species infected. They may block cellular and/or immune defenses against viruses, for example. In some viruses, the function of some of these genes is not understood.

Image

That's enough for one post. In my next post I'll start to cover the replication cycle of coronaviruses.
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Re: A series of posts about virology

Thu Apr 09, 2020 9:48 am

in 1889-1990 a global pandemic of human respiratory disease was described originating in China.

did you mean 1889-1890 ?
 
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Re: A series of posts about virology

Thu Apr 09, 2020 11:10 am

casinterest wrote:
Are you going to do a follow up post about the race for the vaccine? I am interested a bit more in the functionalities involved there. IE what constitutes a "dead" virus? Do they find a way to block the "spike" proteins?

I join.
I believe giving probability estimations or timelines is not the scientist's cup of tea. Let me ask this way:
Doc, are you familiar with the effort of SARS a few years back? How far did the research reach before the virus disappeared?

There are lots of sicknesses without vaccination or cure yet.
Do virologists consider Covid 19 rather tricky or are virologists optimistic that a vaccination or treatment can be found?
Why can't the world be a little bit more autistic?
 
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Re: A series of posts about virology

Thu Apr 09, 2020 4:50 pm

DLFREEBIRD wrote:
in 1889-1990 a global pandemic of human respiratory disease was described originating in China.

did you mean 1889-1890 ?


I did! Oops! Can't edit it now.
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Re: A series of posts about virology

Fri Apr 10, 2020 7:36 pm

Doc, thanks for doing this, very succinct and informative. I question I have had about viral infection in general and in particular this Covid-19. There was a family that was in the news that was nearly wiped out because or being infected, now I understand "existing medical conditions" hastening a patient to crash, but is there any genetic pre-dispostion to the morbidity of this virus, and any others for that matter? And if so, would this explain asymptomatic carriers? One other question, do most virus reproduce by mimicking the "key" of the channel to gain access to the host, or brute force by dissolving a section of the cell wall?
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Re: A series of posts about virology

Sun Apr 12, 2020 4:43 am

The Coronavirus Infection Cycle

Step 1: Attachment

Coronaviruses express an S (spike) protein that protrudes from their surfaces. An example of a spike protein from HCoV-NL63 (an Alphacoronavirus) is shown here:
Image

Each coronavirus binds to a specific receptor on the surface of the target cell. The identity of that receptor is determined by the three-dimensional shape and charge of the spike protein. The spike protein itself is comprised of two domains. One of these domains is the receptor-binding domain that specifically recognizes its receptor. Hidden under that is a second domain called a fusion peptide that can bury itself into the target cell membrane and force the target cell to fuse with the virion. For SARS-CoV and SARS-CoV-2, that surface receptor is the ACE2 protein, which is involved in blood pressure regulating, clotting, immune function, and multiple other functions. Interestingly, HCoV-NL63, whose spike is shown above, not only binds to this same protein, but even to the same surface motif on the protein. In spite of that, genetic sequencing reveals that these two viruses are only distantly related with no sequence or structural homology, suggesting that they independently evolved to bind to the same "hotspot" on the ACE2 surface.

Other coronaviruses bind to different surface receptors on different kinds of cells. Some of them are sugars attached to proteins. But all of these receptor-spike interactions serve the same function: they bind the virus particle to the surface of the cell so that the next steps can occur.

Step 2: Entry

After the S protein binds to the surface receptor, a series of events occur that involve some combination of cleaveage of the S protein by a cell protease and exposure to an acidic pH. The precise sequence of events is different for each CoV, and for SARS-CoV-2 there are still details that have not been worked out.

After binding to ACE2, a serine protease that is expressed on the surface of the target cell called TMPRSS2 cleaves a different spike protein than the one that is bound to the ACE2 molecule. This action reveals the fusion peptide. For SARS-CoV, this action was sufficient to cause fusion of the viral envelope with the cell. There are two hypotheses for the entry of SARS-CoV-2 into cells. The first is that it also fuses at the cell surface. The second is that it is taken into a vesicle called an endosome where a set of proteases called cathepsins help to cleave the protein and activate it. It is debated as to whether acidification of the endosome is required for cell entry. This finding in cell culture raised the possibility that chloroquine or hydroxychloroquine (which inhibit this acidification) might help to block this process, but clinical studies have been disappointing, suggesting that surface fusion may play a significant role.

Whether it is in an endosome or at the cell surface, the fusion domains stab into the host cell membrane and pull the virus particle very close to the cell membrane, squeezing them so close together that they fuse. This releases the nucleocapsid into the cytoplasm (the cytoplasm, or cytosol, is the volume of the cell inside the cell membrane but outside the nucleus).

A diagram of this process (which should be interpreted from the sides to the center step-by-step) is shown here:
Image

Step 3: Expression of Early Viral Genes

The coronavirus genome is called +ssRNA, which stands for "positive-sense single-stranded RNA."

A quick review of 7th grade Biology: when a gene (DNA in cells, RNA in RNA viruses) is read by an RNA polymerase enzyme that makes messenger RNA (mRNA) that can be read by a ribosome, that process is called transcription. When the ribosome binds to this mRNA and uses its instructions to make a protein, that process is called translation. A +ssRNA is an RNA that can serve as an mRNA. That is to say that it can be directly read by a ribosome.

On entry into the host cell cytoplasm, the nucleocapsid proteins that protect the viral RNA are shed and a ribosome binds to the RNA. The first gene to be translated is a very large gene called ORF1 or Rep1a and Rep1b. These two genes overlap and the ribosome has to do a little frameshift between the two, a detail that I'll gloss over here. The proteins encoded by these genes are "polyproteins," a string of proteins that do different things and, like little plastic bits in a model kit, must be broken apart in order to do their work. One of these proteins is a protease and it curls back on itself and clips the other proteins in the chain apart. This protease is of great interest to drug developers who are working to find compounds that might block its action. The proteins produced from this process comprise a set of nonstructural proteins (NSPs), which means that they are proteins that will not be included in the structure of the resulting virion. These processed NSPs then assemble to form what is called the replicase-transcriptase complex. This is an RNA-dependent RNA polymerase (RdRp), which means that it can copy RNA from RNA. No mammal enzyme has this capability and so this enzyme is also an attractive target for drug design. The drugs remdesivir (Gilead) and favipiravir (Fujifilm) are RdRp inhibitors. This complex of proteins copies the viral RNA and in coronaviruses is interesting because it appears to have an error-correcting function that gives coronaviruses a remarkably low mutation rate for RNA viruses. At first, the main function of this protein is to make more mRNAs for the cell's ribosomes to translate so that more virus proteins can be produced.

Other early genes serve to attack cell defenses against viruses. The goals are to stop the cell from expressing its own genes so as to co-opt the cell's resources for viral needs and also to stop the cell from producing molecules like interferon, which block viral replication through a number of mechanisms.

In addition, a portion of rep1a contains a protein that binds to membranes inside the cell. Viral machinery tends to bind to membranes inside the cell so that all of the activity can be focused in a small volume, rather than having pieces of machinery float away.

Step 4: Late Genes, Virion Assembly, and Egress

Later in the infection, the function of the replicase complex switches from producing mRNAs from the viral genome (a two-step process of transcribing a -ssRNA and then re-transcribing it to a +ssRNA) to copying the entire viral genome.

I reviewed above that much of the viral machinery concentrates near intracellular membranes (the cell's endoplasmic reticulum) and indeed the assembly of virions occurs within membranes. Within membranes inside the cell, the various components (RNA bound with N protein, M, E, and S proteins) are assembled in what are called double-membrane-vesicles (where the inner membrane will become the viral envelope). Once they are assembled, they are trafficked through the Golgi complex to the cell membrane and released. A 3D reconstruction by electron microscope tomography of these double-membrane vesicles is shown here:

Image

Alternative Transmission: Formation of Syncytiae

A syncytium is a collection of multiple cells that have fused together to form a single cell with multiple nuclei. In some cases, syncytiae are normal. Muscle cells (heart and skeletal) are syncytiae and also some neurons form a kind of syncytium through a kind of connection called a gap junction. Some viruses, including coronaviruses, can force the formation of syncytiae by expressing their spike proteins on the surface of the infected cell. These can force the cell membrane to fuse with an adjacent cell, which allows the virus to infect that cell without having to bud virions that might be exposed to antibodies.
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Re: A series of posts about virology

Sun Apr 12, 2020 10:07 am

I don't pretend to understand everything, but thank you for the time to explain it. I feel a little bit more knowledgeable and hopeful.

favipiravir
https://www.thepharmaletter.com/article ... ate-avigan

remdesivir

https://www.gilead.com/purpose/advancin ... cal-trials



 basic facts that undermine Trump's briefings

https://www.factcheck.org/2020/04/trump ... onnection/
 
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Re: A series of posts about virology

Sun Apr 12, 2020 9:40 pm

Doc, I have a question about immunity when it comes to viruses.

When you are infected the virus replicated enough that the body' s immune system is alerted to the response and the fighting back is what makes you sick and gives the symptoms. You then over time (usually a few day) develop antibodies to neutralize the virus and memory cells remember the infection for future exposure.

In theory when you contract the same infection again doesn't the replication of the initial virus have to happen to an extent to alert the immune system of the presence of the virus and then the appropriate antibodies are released to neutralize the virus without the host getting sick again. This can explain why someone could test positive for Covid-19 again but not necessarily going to get sick again. Especially given the long incubation time of covid19.

Is this correct or is the immune system respond even faster and if it does, how? Also can an immune person be an asymptomatic carrier even though they will not get sick for however long the immunity lasts.
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Re: A series of posts about virology

Tue Apr 14, 2020 11:01 pm

StarAC17 wrote:
Doc, I have a question about immunity when it comes to viruses.

When you are infected the virus replicated enough that the body' s immune system is alerted to the response and the fighting back is what makes you sick and gives the symptoms. You then over time (usually a few day) develop antibodies to neutralize the virus and memory cells remember the infection for future exposure.

In theory when you contract the same infection again doesn't the replication of the initial virus have to happen to an extent to alert the immune system of the presence of the virus and then the appropriate antibodies are released to neutralize the virus without the host getting sick again. This can explain why someone could test positive for Covid-19 again but not necessarily going to get sick again. Especially given the long incubation time of covid19.

Is this correct or is the immune system respond even faster and if it does, how? Also can an immune person be an asymptomatic carrier even though they will not get sick for however long the immunity lasts.


OK, you just asked a big question and the answer is:

Immunity is not the same thing as antibodies

You can have antibodies that you generated against a virus (like HIV) and yet not be immune to it. If I inject you with an antibody against a virus, it may protect you from that virus and yet you are not immune because when the antibody wears off, you will be susceptible and even those patients who are injected with such an antibody have a lower, but not zero chance of being infected with that virus.

Conversely, it is quite common for children who have been vaccinated for chickenpox to not demonstrate circulating antibodies to the varicella-zoster virus and yet they do not become infected with it.

How can this be?

First, let's talk about how antibodies are created. Antibodies are generated by a type of white blood cell called a B lymphocyte, or a B cell. In the human genome there are three genes that are used to generate antibodies. Within each of these genes, there is a region called the "hypervariable region" that can be rearranged essentially at random (within that region) by certain systems that work in the cell's nucleus. Imagine as an analogy one of those screwdrivers with the exchangeable heads. You can fit eleventy kinds of head onto the same handle. The hypervariable region allows the cell to change the head of the antibody while the rest of the antibody stays the same.

So this randomly-generated hypervariable region of the new antibody is then tested against a bunch of "self" proteins. Antibodies shouldn't react to "self" proteins or that would generate autoimmunity. If it does react to one of those, then the cell is killed ("voted off the island," if you will), which is the result almost all of the time. For the one in several thousand cells that pass this test, then the antibody is tested against a bunch of non-self proteins that are being processed at that time by the immune system and if it binds to one of them, then the cell is allowed to mature and begin to divide so that it can make more antibody. After the infection is cleared, there is a contraction of the number of B cells, but a few are usually kept in reserve somewhere (memory B cells).

We call the presence of antibodies humoral immunity because the antibodies circulate in the plasma (humor). This is important for some viruses, like Hepatitis B, because Hepatitis B Virus must travel through the blood to get to the liver. So when Hepatitis B Virus gets into the blood of someone who is immune, the virus particles are immediately covered in antibodies and taken out of commission. But for a virus like SARS-CoV-2, it only rarely ever gets into the blood and entry into the blood is not essential for infection. Rather, it comes out of the air and infects cells in the respiratory tract. Once a virus is inside a cell, antibodies can't reach it.

So there's a second kind of lymphocyte called a T lymphocyte or a T cell and these cells have a similar set of genes to the antibodies, but the protein they create is called the T cell receptor (TCR).

So on the surface of every cell in your body (except for red blood cells), you express a set of proteins called Major HistoCompatibility (MHC) proteins, sometimes called Human Leukocyte Antigens (HLA). In every cell in your body that has a nucleus (so not red blood cells), proteins are constantly being made. A sample of every protein that is made in the cell is chopped into bits and those little pieces, called polypeptides. A MHC protein is kind of shaped like a hot dog bun with a groove down the middle, and so a little piece of every protein that is being made inside the cell is put into that groove and then the MHC protein with its little polypeptide is presented on the outside of the cell.

Just like B lymphocytes, T cells are constantly being created. They randomly rearrange their TCRs. These TCRs are then challenged against a panel of "self" polypeptides that are being presented on MHC molecules. If they recognize one of those "self" polypeptides, then the cell is negatively selected ("voted off the island"). If they pass this test (and again, only a tiny minority of cells do), then they are tested against the set of foreign peptides being handled by the immune system at that time and if they recognize one of these, then they are allowed to mature.

So when a cell is infected by a virus, the viral proteins start to get produced and just like any other protein being made in the cell, little snippets of viral protein are stuffed into MHC proteins and presented on the surface of the cell. Along comes a T cell that has a TCR that recognizes this MHC-polypeptide combination and when it does, the T cell is activated. It begins to divide so as to make copies of itself. But in addition, it sets off a chain of events directed at killing the infected cell. Signals are sent to the cell that set off a cascade of events called "apoptosis" in which the cell kills itself because a virus cannot replicate in a dead cell. Of course, viruses have systems to block apoptosis, so the T cell also sends out proteins that drill holes in the target cell. After the viral infection is cleared, most of the T cells undergo apoptosis (contraction) just like the B cells, but a few of them are stored away as "memory T cells" for future use should that virus be encountered again.

This kind of immunity is called "cell-mediated immunity." It's much more important for viral infections that infect mucosal surfaces (like the lungs, the GI tract, the nose, the genitourinary tract).

The trouble is that it's easy to test for antibodies. You take a blood sample. You take the antigen against which the antibodies are supposed to react and put it on a surface and then you see if antibodies bind to it. But for an infection like COVID-19, while we will make antibodies, the antibodies really are not likely to be the main important thing in the immune response. It's the cell-mediated response that's important.

So how to test for cell-mediated immunity? Well, every human being (aside from identical twins) has a unique set of MHC proteins called an HLA type. Your T-cells will only respond to cells that display your MHC type. So you'd have to grow cells in culture that have been genetically engineered with the patient's HLA molecules and then infect those cells in culture with the virus and then apply a sample of the patient's T cells to the culture and see if they react, which would have to be done with microscopic immunofluorescence. So you can see that while this is possible, it's incredibly complex and completely impractical in a clinical setting, although a few research studies have used this method.

For patients with SARS, their antibodies against the virus faded in about six months (maximum two years), but I would bet anyone a whole roll of toilet paper that they are still immune, but their immunity is cell-mediated, not humoral. And my guess is that it will be the same for COVID-19.
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StarAC17
Posts: 3799
Joined: Thu Aug 07, 2003 11:54 am

Re: A series of posts about virology

Wed Apr 15, 2020 6:34 pm

DocLightning wrote:
StarAC17 wrote:
Doc, I have a question about immunity when it comes to viruses.

When you are infected the virus replicated enough that the body' s immune system is alerted to the response and the fighting back is what makes you sick and gives the symptoms. You then over time (usually a few day) develop antibodies to neutralize the virus and memory cells remember the infection for future exposure.

In theory when you contract the same infection again doesn't the replication of the initial virus have to happen to an extent to alert the immune system of the presence of the virus and then the appropriate antibodies are released to neutralize the virus without the host getting sick again. This can explain why someone could test positive for Covid-19 again but not necessarily going to get sick again. Especially given the long incubation time of covid19.

Is this correct or is the immune system respond even faster and if it does, how? Also can an immune person be an asymptomatic carrier even though they will not get sick for however long the immunity lasts.


OK, you just asked a big question and the answer is:

Immunity is not the same thing as antibodies

You can have antibodies that you generated against a virus (like HIV) and yet not be immune to it. If I inject you with an antibody against a virus, it may protect you from that virus and yet you are not immune because when the antibody wears off, you will be susceptible and even those patients who are injected with such an antibody have a lower, but not zero chance of being infected with that virus.

Conversely, it is quite common for children who have been vaccinated for chickenpox to not demonstrate circulating antibodies to the varicella-zoster virus and yet they do not become infected with it.

How can this be?

First, let's talk about how antibodies are created. Antibodies are generated by a type of white blood cell called a B lymphocyte, or a B cell. In the human genome there are three genes that are used to generate antibodies. Within each of these genes, there is a region called the "hypervariable region" that can be rearranged essentially at random (within that region) by certain systems that work in the cell's nucleus. Imagine as an analogy one of those screwdrivers with the exchangeable heads. You can fit eleventy kinds of head onto the same handle. The hypervariable region allows the cell to change the head of the antibody while the rest of the antibody stays the same.

So this randomly-generated hypervariable region of the new antibody is then tested against a bunch of "self" proteins. Antibodies shouldn't react to "self" proteins or that would generate autoimmunity. If it does react to one of those, then the cell is killed ("voted off the island," if you will), which is the result almost all of the time. For the one in several thousand cells that pass this test, then the antibody is tested against a bunch of non-self proteins that are being processed at that time by the immune system and if it binds to one of them, then the cell is allowed to mature and begin to divide so that it can make more antibody. After the infection is cleared, there is a contraction of the number of B cells, but a few are usually kept in reserve somewhere (memory B cells).

We call the presence of antibodies humoral immunity because the antibodies circulate in the plasma (humor). This is important for some viruses, like Hepatitis B, because Hepatitis B Virus must travel through the blood to get to the liver. So when Hepatitis B Virus gets into the blood of someone who is immune, the virus particles are immediately covered in antibodies and taken out of commission. But for a virus like SARS-CoV-2, it only rarely ever gets into the blood and entry into the blood is not essential for infection. Rather, it comes out of the air and infects cells in the respiratory tract. Once a virus is inside a cell, antibodies can't reach it.

So there's a second kind of lymphocyte called a T lymphocyte or a T cell and these cells have a similar set of genes to the antibodies, but the protein they create is called the T cell receptor (TCR).

So on the surface of every cell in your body (except for red blood cells), you express a set of proteins called Major HistoCompatibility (MHC) proteins, sometimes called Human Leukocyte Antigens (HLA). In every cell in your body that has a nucleus (so not red blood cells), proteins are constantly being made. A sample of every protein that is made in the cell is chopped into bits and those little pieces, called polypeptides. A MHC protein is kind of shaped like a hot dog bun with a groove down the middle, and so a little piece of every protein that is being made inside the cell is put into that groove and then the MHC protein with its little polypeptide is presented on the outside of the cell.

Just like B lymphocytes, T cells are constantly being created. They randomly rearrange their TCRs. These TCRs are then challenged against a panel of "self" polypeptides that are being presented on MHC molecules. If they recognize one of those "self" polypeptides, then the cell is negatively selected ("voted off the island"). If they pass this test (and again, only a tiny minority of cells do), then they are tested against the set of foreign peptides being handled by the immune system at that time and if they recognize one of these, then they are allowed to mature.

So when a cell is infected by a virus, the viral proteins start to get produced and just like any other protein being made in the cell, little snippets of viral protein are stuffed into MHC proteins and presented on the surface of the cell. Along comes a T cell that has a TCR that recognizes this MHC-polypeptide combination and when it does, the T cell is activated. It begins to divide so as to make copies of itself. But in addition, it sets off a chain of events directed at killing the infected cell. Signals are sent to the cell that set off a cascade of events called "apoptosis" in which the cell kills itself because a virus cannot replicate in a dead cell. Of course, viruses have systems to block apoptosis, so the T cell also sends out proteins that drill holes in the target cell. After the viral infection is cleared, most of the T cells undergo apoptosis (contraction) just like the B cells, but a few of them are stored away as "memory T cells" for future use should that virus be encountered again.

This kind of immunity is called "cell-mediated immunity." It's much more important for viral infections that infect mucosal surfaces (like the lungs, the GI tract, the nose, the genitourinary tract).

The trouble is that it's easy to test for antibodies. You take a blood sample. You take the antigen against which the antibodies are supposed to react and put it on a surface and then you see if antibodies bind to it. But for an infection like COVID-19, while we will make antibodies, the antibodies really are not likely to be the main important thing in the immune response. It's the cell-mediated response that's important.

So how to test for cell-mediated immunity? Well, every human being (aside from identical twins) has a unique set of MHC proteins called an HLA type. Your T-cells will only respond to cells that display your MHC type. So you'd have to grow cells in culture that have been genetically engineered with the patient's HLA molecules and then infect those cells in culture with the virus and then apply a sample of the patient's T cells to the culture and see if they react, which would have to be done with microscopic immunofluorescence. So you can see that while this is possible, it's incredibly complex and completely impractical in a clinical setting, although a few research studies have used this method.

For patients with SARS, their antibodies against the virus faded in about six months (maximum two years), but I would bet anyone a whole roll of toilet paper that they are still immune, but their immunity is cell-mediated, not humoral. And my guess is that it will be the same for COVID-19.


Ok so with "cell-mediated immunity." which makes sense for a respiratory virus as the blood is not infected. I understand that and that would make sense that some people who recovered might not have antibodies in their blood when tested for them.

If someone who got and recovered from COVID19 still be able to test positive again and potentially be contagious, given the long incubation time and respiratory nature of this virus. With most viruses you aren't contagious until your sick and there is a much shorter incubation period.
Also on the second exposure wouldn't the virus be replicating in the body (as it did initially) enough to set the alarm bells of the immune system off for those memory T-cells to kick in.

If that is the case then it would make sense that someone could test positive again but not at a risk to get sick from it as the symptoms at the end of the day are the body fighting back.
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