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Team:PYMS GZ China/Description


Description
Background

The respiratory illness COVID-19, caused by SARS-CoV-2, has spread to countries across the world. Many theorize that the progenitor of SARS-CoV-2 transferred into humans through human-to-animal interaction. In March, the World Health Organization announced that COVID-19 is a global pandemic. In addition, COVID-19 is highly infectious, spreading from person to person.

It is highly recommended for individuals to wear masks and social distance, as respiratory droplets can travel through the air, entering the nose and mouth. Symptoms can range from mild (asymptomatic) to severe illness that cause death. Some key symptoms are fever, cough, difficulty breathing, loss of smell or taste, sore throat, nausea or vomiting, headache, and chest pain. Those at greater risk for severe illness include older adults and those with underlying medical conditions, such as asthma, a weakened immune system, or serious heart conditions.

Human ACE2

The angiotensin-converting enzyme 2 (ACE2) plays an important role in allowing the virus to infect human cells; it is the entry point of SARS-CoV-2. ACE2 is a surface protein that is present in many human cells and tissues such as the lungs, heart, blood vessels, kidneys, liver, and gastrointestinal tract. ACE2 is key in regulating processes such as blood pressure, inflammation, and healing wounds. ACE2 is present in high amounts in type 2 pneumocytes that are present in alveoli, the location where oxygen is taken in and carbon dioxide is expelled. ACE2 has an important role in the biochemical pathway because it regulates the protein angiotensin II (ANG II) that harms blood vessel linings and various tissues through increasing blood pressure and inflammation. More specifically, ACE2 transforms ANG II into other molecules that neutralize the negative effects of ANG II.

RBD of Spike Protein

The Spike protein on the SARS-CoV-2 contains a segment called the receptor binding domain (RBD) that recognizes and binds to the host receptors, ACE2, on human cells. It has been found that the RBD of Spike protein of the SARS-CoV-2 has a higher binding affinity to ACE2 than previous viruses. When the S-protein found on the SARS-CoV-2 virus binds to the ACE2, the virus is able to enter and infect the host cell. By infecting the cell, the virus hijacks the host cell machinery for its reproduction, and causes inflammation and death of alveoli cells, whose function is important in supplying oxygen to the body, leading to damage in the lungs and other organs.

HEK 293

The HEK293 cells are human embryonic kidney cells. They are very easy to grow and maintain. Oftentimes, they are used in labs due as they are easy and susceptible to transfection.

PCR Mutagenesis

Polymerase chain reaction (PCR) mutagenesis is used for generating site-directed mutations. Mutagenesis is the creation of genetic mutations in DNA. Primers are designed to edit the original strain of DNA by not completely matching the template strand. This allows the sequence of DNA nucleotides to be altered. PCR mutagenesis allows for the creation of millions of copies of the modified DNA. PCR consists of three steps: denaturation, annealing, and elongation. In our project, we used PCR mutagenesis to create our S variant constructs. We created two different S variants- the original wildtype strain and the highly infectious G614D.

Pseudovirus

We used a highly efficient lentiviral-based pseudovirus system for assaying S-RBD and ACE2 interaction, therefore infectivity and immunity. A pseudovirus lacks viral virulent components which eliminate the possibility of an active infection to an exposed individual, yet allow it to be handled in a low risk biosafety level (BSL) level 2 lab. In addition, a pseudovirus can simulate the RBD-ACE2 interaction and can be quantified by a reporter assay, allowing infectivity to be precisely measured.

Project Inspiration

Our team is from all around the world - from California to Massachusetts, and halfway around the globe in China. With many of us residing in California, the state with the most COVID-19 cases in the country, life is still far from normal. In other areas, a sense of normalcy has been restored as they are allowed to attend in person school and engage in other extracurricular activities with their peers. With the introduction of online school and the ever climbing number of cases, our all-girls team knows that the battle against the virus is still raging on and it will be for a long time. This realization inspired our team to try to be a part of the solution. Changes to our normal routines and the Stay-At-Home orders left us with some flexible time which we were motivated to use in an impactful way. Seeing many people we know ignore the mask mandates and think SAR-CoV-2 was not real, we were motivated to spread awareness of the severity of the situation. Upon researching the information on Sars-CoV-2, our literature review revealed a weakness in the research being done- there was no easy way to test the infectivity of the different strains of SARS-CoV-2. Knowing the infectivity of the different strains is crucial in guiding future research to combat the virus, we were inspired to focus our project on creating a universal assay system using a pseudovirus to test the infectivity and immunity of current and future strains of SARS-CoV-2.

References
  1. “Immune Responses and Immunity to SARS-CoV-2.” European Centre for Disease Prevention and Control, 16 July 2020, www.ecdc.europa.eu/en/covid-19/latest-evidence/immune-responses. 
  2. RnD Systems. “ACE-2: The Receptor for SARS-CoV-2.” Www.rndsystems.com, www.rndsystems.com/resources/articles/ace-2-sars-receptor-identified.
  3. Saplakoglu, Yasemin. “Coronavirus 'Spike' Protein Just Mapped, Leading Way to Vaccine.” LiveScience, Purch, 19 Feb. 2020, www.livescience.com/coronavirus-spike-protein-structure.html.
  4. Sriram Postdoctoral Fellow, Krishna, et al. “What Is the ACE2 Receptor, How Is It Connected to Coronavirus and Why Might It Be Key to Treating COVID-19? The Experts Explain.” The Conversation, 6 Sept. 2020, 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.
  5. Shang, Jian, et al. “Cell Entry Mechanisms of SARS-CoV-2.” PNAS, National Academy of Sciences, 26 May 2020, www.pnas.org/content/117/21/11727. 
  6. The Francis Crick Institute. “Structural Analysis of COVID-19 Spike Protein Provides Insight into Its Evolution.” ScienceDaily, ScienceDaily, 9 July 2020, www.sciencedaily.com/releases/2020/07/200709105122.htm.