Disclaimer: I am not a physician or a virologist. I have a PhD in cell and molecular biology. I did research in medical schools and was part of a clinical trial in a biopharma company. I have grown several viruses and have performed all of the laboratory tests described in this blog.

SUMMARY: Two different tests are required to trace COVID-19 disease. One test looks for viruses, indicating active disease. A different test determines if a person has recovered from the disease by searching for antibodies to the virus.

TESTS FOR ACTIVE DISEASE must detect either the viral genome or the Spike protein. Spike in pink and RNA in blue.

Can these tests be done at home? Pregnancy tests can be done at home, but similar reagents specific for coronavirus are not reliably available at this time.

Can these tests be done quickly in a doctor’s office? Some rapid tests are offered in a doctor’s office, but others must be sent to a lab for more specific testing. Some rapid tests do not have a high accuracy or reliability. They can confirm clinical symptoms, but may not detect low levels of virus in a person. See below RDT (Rapid Diagnostic Tests).

What confirms the presence of coronavirus with or without symptoms? Currently two tests are used. A Genomic test searches for the viral RNA; an Antigen test searches for the Spike protein. The diagram shows internal RNA and external Spikes that bind the virus to lung cells.

Genomic test for SARS-CoV-2 Virus. We see TV crime shows where a drop of blood reveals the identity of the killer in minutes. It works, but just takes more time. First, more copies of the blood DNA must be made. The enzyme DNA polymerase goes back and forth over the DNA chains and makes millions of copies. This is called PCR (polymerase chain reaction) and requires several hours before DNA tests can be performed.

Viral Genomic testing requires one additional testing step. Coronaviruses have RNA, not DNA as genetic material. Viral RNA must first be turned into DNA. RT (reverse transcriptase) turns RNA into DNA and then PCR makes all the copies. This paired test called RT-PCR is extremely accurate. Why is RT-PCR necessary? The viral genome is small. Many copies must be made to be detected in the test. The amount of virus a person has does not indicate how sick they are.  Some people breathe out lots of virus, but show no symptoms of being ill. Some people are very sick with low levels of virus.

Antigen test for Spike protein on SARS-CoV-2 Virus.  Antibodies to Spike proteins are bound into tiny wells of plastic testing plates. Saliva or nasal swab material is added to the wells. If virus is present in the material, it will stick to the Spike antibodies and register as a positive test.

TESTING FOR RECOVERY FROM COVID-19. What happens when people get well? During COVID-19 disease, the immune system makes antibodies to get rid of the virus. Antibodies are also called immunoglobulins (Ig), and we humans have five different types. Within a few days fighting a disease, the immune system makes IgM. One to three weeks after being ill, white blood cells called B cells make IgG or gamma globulin. Some of these B cells hopefully become Memory B cells. Immunity means you cannot get sick from the virus again.

How should immunity work? Measles vaccine was developed to give lifetime immunity. If you received the vaccine as a baby, there are memory B cells to the virus in your body today. If you are exposed to measles when you are fifty years old, the B cells will rush from their sequestered hiding to start a new assault and destroy the measles virus before it can make you ill.

How do you test for antibodies or IgG?  A portion of the Spike surface protein named S1 seems to create the largest immune response. This protein S1 is used in a test called ELISA (enzyme-linked immunosorbent assay). This is the reverse of the test to detect the antigen Spike. Using plastic plates with small wells, the S1 protein is stuck to the bottom of the wells. Then patient serum is added to the wells. If there is IgG to the S1 protein, they will stick together. Several dilutions of the serum will register as a high or low immune response. This test takes several days in a laboratory, but is the best test to determine if you have antibodies.

RDT: How do the rapid tests work? The RT-PCR and ELISA tests are the standards to shorten. Most testing is proprietary, but RT-PCR can be shortened by using a smaller piece of the RNA. A stick antigen test is set up like a pregnancy test. The antibodies to Spike S1 are on the stick. As the nasal fluids flow down the stick, any virus particles will stick and register as a color band. The reverse is again used to test for IgG in recovery. Spike S1 is stuck to the stick. As blood serum flows on the stick, IgG will stick to the Spike S1 proteins.

Does recovery from COVID-19 provide immunity? No one knows.  It is not known if a mild case or a severe case provides the same immunity. And if antibodies are produced, no one knows how long they last.

 How long do people have detectable virus?No one knows. Some people shed (breathe out) virus for months and feel fine. Normally within two weeks the disease is over, but some have symptoms for months.

Has the virus mutated? Surveys indicate that the European-United States virus strain had a Spike protein mutation back in February-March and is the dominant strain in the US today. This mutation was a single amino acid change that may cause the virus to bind more easily to lung receptors, but does not change any of the antigen or antibody tests.

Disclaimer: I am not a physician or a virologist. I have a PhD in cell and molecular biology. I did research in medical schools and was part of a clinical trial in a biopharma company. I have grown several viruses, but none in the coronavirus family. I am not associated with any group making vaccines to SARS-CoV-2. I have performed most of the laboratory tests described in this blog to detect virus and antibodies. I have made monoclonal antibodies, but not for COVID.

Summary: Currently over one hundred candidates for a vaccine against SARS-CoV-2 are in development. The best ones will provide lasting immunity, which is lifetime resistance to disease. Because Pfizer and Moderna have announced results, I will lead with their novel vaccine developments.

What is a messenger RNA (mRNA) vaccine and why is it different? In the previous post, this virus was followed as its mRNA entered a lung cell and made copies of itself. That is exactly how the mRNA vaccines work. However, be assured several modifications are used in the mRNA injected as vaccines. First, the complete virus RNA is NOT injected as a vaccine. The complete RNA for this virus is known, but both vaccines only use a section of the RNA that codes for the Spike surface protein. To further protect you and to create a greater immune response, a synthetic copy of the mRNA is made in the lab. The Pfizer mRNA is called BNT162b2; Moderna is mRNA-1273.

What else is in the vaccine? RNA is a very fragile molecule. In the lab, gloves have to be worn and glassware is baked at 350 ͦ F for 2 hours. RNA can fall apart in seconds. The vaccines use lipids to stabilize the mRNA, but cold is still required. The Pfizer vaccine mRNA is formulated with lipids as the mRNA–lipid nanoparticle drug product. It requires -100 ͦ F or dry ice for storage. Moderna coats its mRNA with an ionizable (cationic) lipid; cholesterol; DSPC (phospholipid) and PEG2000-DMG (conjugated anti-aggregation lipid). This combination permits regular freezer and even refrigerator storage of the vaccine for a short time.

How does the mRNA vaccine create immunity? When the vaccine is injected into the muscle, it is noticed as a foreign substance by muscle cells and several immune cells. Blood cells notice and react. There are immune cells in the skin and muscles called dendritic cells. These cells take up the mRNA and like lung cells, they use the mRNA and start making spike proteins. Spike proteins are REALLY noticed as foreign and a whole cascade of immune events begin. It appears that one dose of the vaccine is not enough to cause a full response. However, within days of a second injection, a full blown immune reaction begins. The response is such that 95% of those given a second injection are protected from COVID or at least get a mild case. Spike is a good antigen to invoke immune response.

How have vaccines been made through time and what are the new strategies being used for COVID protection? Vaccines mimic natural infection of a virus or bacteria. The vaccine must be at a low enough dose to not create a full blown illness or use a modified virus or bacteria to create a response that protects from the dangerous pathogen.

How does immunity to a virus naturally happen? Let’s use measles as an example. When measles enters your body, it is recognized as foreign mainly because of two envelope knobs known as H and F. Immune cells in your body take in viruses and start a response that ends with B white blood cells making antibodies to neutralize the measles viruses. This is one of the tests used for vaccine efficacy—production of neutralizing or virus-killing antibodies. To have lasting immunity, some of the B cells convert to Memory B cells that last the rest of your life. This sequence takes about three weeks. If you confront measles viruses in the future, Memory B cells will come out of hiding and within days make lots of antibodies that will bind viruses and help destroy them before the infection can make you ill again.

How can a vaccine replicate those events?

1. Use a similar virus. Some of the first vaccinations were like Jenner’s use of cowpox virus to give protection to the similar smallpox virus.
2. Use small amounts of virus. This will give a response without disease, like cowpox, but is no longer used.
3. Use a virus inactivated or fixed with formalin. The virus will be intact and recognized as foreign, but will not be able to cause disease because it is dead. Pertussis is an example as are Salk polio vaccines.
4. Use an attenuated (A Ten you A ted) virus. Viruses are grown in chicken embryos or cell cultures. Once viruses adapt to grow in non-human cells, they will no longer grow in human cells. The measles, mumps and rubella MMR trio are attenuated as are chicken pox, influenza, yellow fever and oral polio vaccines.
5. Use a surface protein, like Spike. Antigens, like Spike, can be purified for a vaccine. Spike could even be changed slightly. Spike particles would not be infectious, but enough of them, would cause a response. The Spike protein could also be made synthetically to not have any virus involved. The drawback would be viral mutations changing Spike so new viruses would not be recognized. The Hepatitis B surface antigen and HIB b (Haemophilus influenza b) that causes infant meningitis are antigen vaccines.
6. Build an artificial nanoparticle to mimic a virus. Bill Gates is funding such a project. An artificial nano particle with lots of sides is built by machines. It would take a billion nano particles to go end to end on your meter or yardstick. So really, really, small. The final step is to decorate the nanoparticle with Spike pieces so it looks like a porcupine. Ordered arrays are most antigenic. This can be given as a vaccine without any live genetic material. One fantastic advantage in development thus far is its stability in heat.
7. Use an adeno virus to transport viral RNA turned into DNA. Adeno viruses cause common colds in humans and our genetic relatives, chimpanzees and gorillas. At least 26 human adeno variants are known. There have been problems with some adeno viruses previously used as vectors, but lots of research has helped that. This virus RNA for spike converted to DNA is placed into the virus.  Vaccine is injected or used as a nasal spray. Again Spike is made in response and you develop an immune response. These vectors are used by Astrazeneca with Oxford and a similar vector used by Johnson&Johnson.

SUMMARY.  Vaccines usually take years to develop. This is not just the design phase, but the testing phase. The ultimate tests of a vaccine are how long does immunity last and are you completely protected from the disease.

Disclaimer: I am not a physician or a virologist. I have a PhD in cell and molecular biology. I did research in medical schools and was part of a clinical trial in a biopharma company. I have grown several viruses, but none in the coronavirus family. I am not associated with any group making vaccines to SARS-CoV-2. I have performed most of the laboratory tests described in this blog to detect virus and antibodies. I have made monoclonal antibodies, but not for COVID.

Do SARS CoV-2 viruses have to enter your lungs before you get sick? Yes. This section uses one virus and one cell to illustrate viral entry. But how many viruses will make you sick? I will now use this virus and COVID for the disease.  

How many SARS CoV-2 viruses do you have to breathe in before you are sick with COVID-19? I found this information on https://www.erinbromage.com/post/the-risks-know-them-avoid-them   It has been proposed that one thousand (1,000) virus particles can give you COVID. Viruses can be from different people at different times. Some investigators think the more particles you take in, the worst your disease, but this has not been proven. However, someone with severe disease releases more virus and greatest amounts are released on days 3-5 of disease.

How many particles are in the air from breathing, talking and coughing/sneezing? Air from the nose and mouth is made of respiratory droplets. Droplets can be large or small. You can even see a big sneeze in the air. Most large droplets quickly fall to the ground (where the kids are), but small ones hang in the air. Normal breathing release twenty virus particles in the air each minute, so 50 minutes near him/her will infect you. Talking releases two hundred viruses a minute, so infecting you in five minutes. A cough or a sneeze can release up to 200,000,000 virus particles that can fill a room/restaurant.

How does this virus enter your cells? A virus particle enters a cell using a lock-and-key mechanism. The key on the outside of the virus is the Spike protein. The lock is the ACE-2 receptor. This receptor named Angiotensin-converting enzyme 2 is an enzyme attached to cells located in the lungs, heart, kidney, and intestines. ACE-2 lowers blood pressure. Once Spike is attached to ACE-2, the membrane door opens and allows the virus to enter the cell, and blood pressure control is lost.

What parts of the virus enter the cell? As the complete virus is entering, the outside membrane comes off, like taking off an overcoat. This releases the strand of RNA (in blue) that is its genetic material. This RNA form is called positive sense or +RNA for a virus. This is the same form of RNA you have called mRNA for messenger RNA.

How do your cells normally use mRNA? For example, how does a cell make insulin?  Your DNA gene for insulin is in the nucleus (and stays there). An mRNA copy of the gene leaves the nucleus and the cell machinery translates the message into insulin. Multiple mRNAs can be used at the same time to make thousands of insulins.

What happens to the viral +RNA inside your lung cell? The virus has two goals: to make more copies of the +RNA and to make proteins from it, like spike. There is no thinking involved, these are automatic events based on the biology of the molecules present. Your lung cell will start making viral proteins from the +RNA, just as if it were one of your own mRNAs. The lung cell cannot tell that it is making something harmful. The virus has hijacked your cell. So as the viral proteins are being made, what is going on with the +RNA?

How does this virus make more copies of itself? Each virus has a tricky way to make copies of RNA. In the last post I said I was going to explain mechanisms of virus replication in cells, like going from RNA to DNA. However, that pathway is not necessary for SARS CoV-2 to replicate in human cells. I decided to switch to more information about this virus after Pfizer released their vaccine data. The Pfizer vaccine inoculates people with synthetic viral +RNA. In the next post the details will be given, but the RNA injected is used as mRNA just like in the lung infection.

This virus makes a –RNA copy. Why? Assume only one +RNA entered your lung cell. One of the tricky viral proteins made from the original +RNA is an enzyme that will start making –RNA strands using the +RNA as a template. The –RNA cannot be used as mRNA, however, yet another viral enzyme will go backwards to make LOTS of +RNA copies, using –RNA copies as templates. These new +RNA can be used as mRNA or to make new viruses. Parallel mechanisms are making all the parts for new virus particles—new +RNA and the parts like Spike.

How do the pieces of virus form actual virus? Viruses are unique because they can self-assemble very quickly. When the lung cell cytoplasm is filled with millions of the individual components listed above, in seconds they come together into new virus particles. Each new virus is a replica of the original virus.

How do the new viruses leave the cell? Once assembled, viruses leave the lung cell through the cell membrane. Within hours of infection, thousands of new viruses infect more lung cells. Many cells break apart and die. This is called lysis and it releases cell parts that can cause immune responses like fluid accumulation and inflammation in the lungs. Some cells stay alive, making more and more viruses. As virus spread to more cells, the response overwhelms the lungs.

Can the lungs recover from this viral infection? When human lungs are infected with just a few coronaviruses, the immune system can destroy them and the person can survive. As the viral load increases, the lungs can become inflamed which involves fluid and cell molecules called cytokines that can also cause more fluid and cellular responses or pneumonia can develop. You may have heard of cytokines like interferon and interleukin in cancer therapy. As the lungs fill with fluid, ventilation may be needed. The problem with the response is that the lungs are fragile and the pressure needed to increase oxygen into the lungs can result in damage to the cells. All of this time more lung cells are infected and falling apart. This is a deadly virus.

Diagram from free article: https://theconversation.com/what-is-the-ace2-receptor-how-is-it-connected-to-coronavirus-and-why-might-it-be-key-to-treating-covid-19-the-experts-explain-136928

The next post, in a few days I promise, will be about vaccines.