Reverse engineering the coronavirus (SARS-CoV-2)

Start here: corona.py

💭 Background

This project applies techniques from reverse engineering to understand the SARS-CoV-2 virus. The goal here is simply to build an understanding of the virus from first principles.

Biology vs. software

Biological systems are fundamentally information processing systems. While not a perfect analogy, software provides a useful framework for thinking about biology. The table below provides a rough outline of this analogy.

🔬 Biology	💻 Software	Notes
nucleotide	byte
genome	bytecode
translation	disassembly	3 byte wide instruction set with arbitrary "reading frames"
protein	function	a polyprotein is a function with multiple pieces
protein secondary structure	basic blocks	80% accuracy in prediction
protein tertiary structure		This seems like the hard one to predict: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0205819
quaternary structure	compiled function with inlining	https://en.wikipedia.org/wiki/Protein%E2%80%93protein_interaction_prediction
gene	library	bacteria are statically linked, viruses are dynamically linked
transcription	loading
protein structure prediction	library identification
genome analysis	static analysis
molecular dynamics simulations of protein folding	dynamic analysis	Simulation doesn't seem to work yet. Constrained by tooling and compute.
no equivalent	execution	We are reverse engineering a CAD format. Runs more like FPGA code, all at once. No serial execution. (What are the FPGA reverse engineering tools?)

🔧 Progress

Downloading the SARS-CoV-2 genome

GenBank is the NIH genetic sequence database, an annotated collection of all publicly available DNA and RNA sequences. The SARS-CoV-2 sequences available in GenBank have been downloaded in download_sequences.py.

Translating RNA to proteins

lib.py contains a function translate that converts an RNA sequence to a chain of amino acids. This function is used in corona.py.

Annotating functions

The translate function is used in corona.py to identify and annotate functions for all proteins encoded by the genome.

Folding proteins

The OpenMM toolkit is used for molecular simulation of protein folding in fold.py.

💡 Work to be done

• Automatic extraction of genes from different coronaviruses
• Good multisequence compare tool
• Molecular dynamics?
• Secondary Structure prediction on orf1a?

❓ Open questions

• How is orf1ab cleaved into polypeptides? Can we predict this from the sequence?
• How do the researchers know (guess?) where orf1ab cleaves?
  • nsp3 and nsp5 do it -- https://www.pnas.org/content/pnas/103/15/5717.full.pdf
• Which protein is the immune system responding to?
  • "spike" and "nucleocapsid" -- http://www.cmi.ustc.edu.cn/1/3/193.pdf
  • Are some people already immune from exposure to other coronavirus?
• Find the "furin cleavage site" in the "spike glycoprotein"
  • It might be at the "PRRA" -- https://www.sciencedirect.com/science/article/pii/S0166354220300528
  • Use ProP or PiTou to predict? -- https://en.wikipedia.org/wiki/Furin
• How similar are the other coronaviruses? (causes colds, not either SARS or MERS)
  • alpha
    • https://en.wikipedia.org/wiki/Human_coronavirus_229E (simpler, though targets APN)
    • https://en.wikipedia.org/wiki/Human_coronavirus_NL63 (targets ACE2!)
      • https://www.ncbi.nlm.nih.gov/nuccore/MG772808
  • beta
    • https://en.wikipedia.org/wiki/Human_coronavirus_OC43 (targets Neu5Ac)
      • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2095096/pdf/JIDMM17330.pdf
      • Specifically, how similar is the N protein OC43, SARS v1, and SARS v2?
    • https://en.wikipedia.org/wiki/Human_coronavirus_HKU1 (targets Neu5Ac)
    • MERS-CoV
    • SARS-CoV
    • SARS-CoV-2
• What adds the phosphate group to the N protein? Kinase?

💧 Testing

How tests work

• All based on https://en.wikipedia.org/wiki/Reverse_transcription_polymerase_chain_reaction
• USA -- https://www.fda.gov/media/134922/download
  • selected from regions of the virus nucleocapsid (N) gene
  • 28286---28308--28332---28358
  • 29163---29187--29210---29230
  • https://biosearchtech.a.bigcontent.io/v1/static/coa_KIT-NCOV-PP1-1000_Lot-No-143503
• South Korea -- http://www.kogene.co.kr/eng/about_us/news/listbody.php?h_gcode=board&h_code=7&po_no=288
  • E gene detection (same for all coronavirus)
  • specific RdRp detection

Homemade test?

• Isolation of viral RNA (no matter what)
  • https://www.qiagen.com/us/products/diagnostics-and-clinical-research/sample-processing/qiaamp-viral-rna-mini-kit/#orderinginformation
• Primers and probes (to detect SARS-CoV-2)
  • https://www.biosearchtech.com/products/pcr-kits-and-reagents/pathogen-detection/2019-ncov-cdc-probe-and-primer-kit-for-sars-cov-2
  • Wouldn't need if using a nanopore sequencer (nanopore MinION)
• RT-qPCR Master Mix (to PCR)
  • https://www.thermofisher.com/order/catalog/product/A15300#/A15300
  • Probably wouldn't need if using a nanopore sequencer
• All in one?
  • https://www.chaibio.com/coronavirus
  • Open qPCR, understand https://www.chaibio.com/openqpcr
  • FAM and HEX fluorophores?

💊 Possible treatments and prophylactics

⚠️ Disclaimer: The information in this repository is for informational purposes only. It is not medical advice.

Hydroxychloroquine + zinc

• Zinc blocks RdRp
• https://jvi.asm.org/content/91/21/e00754-17 -- how similar is Hep E RdRp?
• https://www.ncbi.nlm.nih.gov/pubmed/21079686
• Chloroquine Is a Zinc Ionophore (allows zinc into the cell)
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4182877/
• Without zinc (seems ineffective)
  • https://www.recoverytrial.net/files/hcq-recovery-statement-050620-final-002.pdf (where is study?)
• With zinc
  • https://www.infezmed.it/media/journal/Vol_28_2_2020_9.pdf
  • https://www.sciencedirect.com/science/article/pii/S0306987720306435
  • No results yet: https://www.clinicaltrials.gov/ct2/show/NCT04377646

RdRP inhibitors

• Favipiravir (prodrug for favipiravir-RTP)
• Adenosine Analog
  • Remdesivir (prodrug for GS-441524)
    • https://www.nejm.org/doi/pdf/10.1056/NEJMoa2007764?articleTools=true
  • Galidesivir

Dexamethasone

• corticosteroid = anti-inflammatory + immunosuppressant
• https://www.medrxiv.org/content/10.1101/2020.06.22.20137273v1.full.pdf
• Effects in late stage (blocking immune response?)

Lopinavir-Ritonavir (AIDS cocktail)

• Both drugs are protease inhibitors
• Wouldn't expect it to work, don't think covid has protease
• No effect: https://www.recoverytrial.net/files/lopinavir-ritonavir-recovery-statement-29062020_final.pdf

📚 Resources

Coronavirus-related publications

• Chapter 4 - Coronavirus Pathogenesis -- https://www.sciencedirect.com/science/article/pii/B9780123858856000092
• https://www.futuremedicine.com/doi/pdf/10.2217/fvl-2018-0008
• https://www.sciencedirect.com/science/article/pii/S2095177920302045
• http://korkinlab.org/wuhan
• https://github.com/mattroconnor/deep_learning_coronavirus_cure

Biology

• textbooks
  • Molecular Biology of the Cell
• classes
  • better tests - https://ocw.mit.edu/courses/biology/7-012-introduction-to-biology-fall-2004/index.htm
  • suspected better lectures - https://ocw.mit.edu/courses/biology/7-014-introductory-biology-spring-2005/index.htm
  • alternative lectures - https://youtube.com/playlist?list=PLGhmZX2NKiNldpyRUBBEzNoWL0Cso1jip
  • basics - https://www.khanacademy.org/science/biology

Bioinformatics

• Nextstrain
  • https://nextstrain.org/ncov
  • https://github.com/nextstrain/ncov
  • https://github.com/nextstrain/augur
• https://biopython.org/
• https://github.com/cogent3/cogent3
• https://www.reddit.com/r/bioinformatics/comments/191ykr/resources_for_learning_bioinformatics/
• https://en.wikipedia.org/wiki/Bioinformatics

Epidemic modeling

• https://gabgoh.github.io/COVID/index.html
• http://epidemicforecasting.org

Antibodies

• https://www.medrxiv.org/content/10.1101/2020.07.09.20148429v1.full.pdf
• https://www.medrxiv.org/content/10.1101/2020.05.11.20086439v2.full.pdf

Masks

• Looking for large controlled studies measuring infection rate with/without wearing mask
• https://bmjopen.bmj.com/content/bmjopen/5/4/e006577.full.pdf -- cloth masks bad surgical masks nothing
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2662657/pdf/08-1167_finalRCME.pdf -- unclear, low adherence
• https://sci-hub.tw/10.1001/archpedi.1987.04460060111049 -- masks + goggles help, unclear on mask type
• https://onlinelibrary.wiley.com/doi/epdf/10.1111/j.1750-2659.2011.00198.X -- N95 ~2x more effective than medical masks
• https://jamanetwork.com/journals/jama/article-abstract/184819 -- N95 and surgical similar in effectiveness

Vaccines

• https://en.wikipedia.org/wiki/COVID-19_vaccine
• https://www.nytimes.com/interactive/2020/science/coronavirus-vaccine-tracker.html
• generic vaccine types -- https://www.niaid.nih.gov/research/vaccine-types
• Nucleic Acid Vaccines (none are currently used)
  • Use mRNA to produce viral proteins
    • "It's a very unique way of making a vaccine and, so far, no (such) vaccine has been licenced for infectious disease"
    • Moderna, mRNA-1273 -- https://clinicaltrials.gov/ct2/show/NCT04283461
    • https://www.biorxiv.org/content/10.1101/2020.04.22.055608v1.full.pdf
    • https://www.medrxiv.org/content/10.1101/2020.06.30.20142570v1.full.pdf
  • DNA vaccine
    • "No DNA vaccines have been approved for human use in the United States"
    • Based on injecting DNA (plasmid) that expresses the spike protein
    • https://siasky.net/bACLKGmcmX4NCp47WwOOJf0lU666VLeT5HRWpWVtqZPjEA
  • Viral Vector Vaccines
    • "There are no viral vector vaccines currently on market for use in humans."
    • adenovirus vector, AZD1222
    • recombinant adenovirus type 5 vector, Ad5-nCoV
• Protein-Based Vaccines (subunit vaccines?)
• Whole-Virus Vaccines (inactivated vs live-attenuated)
  • Like old vaccines (live attenuated?)
  • In phase 3 already, Sinopharm = 15,000 people
• Nanoparticle vaccine?
  • https://www.ipd.uw.edu/2019/03/ipds-first-nanoparticle-vaccine/
• Reverse Engineering
  • https://berthub.eu/articles/posts/reverse-engineering-source-code-of-the-biontech-pfizer-vaccine/
  • https://berthub.eu/articles/posts/part-2-reverse-engineering-source-code-of-the-biontech-pfizer-vaccine/

Genome studies (what genes = bad covid)

• https://www.nejm.org/doi/full/10.1056/NEJMoa2020283?source=nejmtwitter&medium=organic-social

DNA Synthesis

• Produce "oligos" of around 170-200 bp
  • "Phosphoramidite chemistry" -- it's a chemical process
  • Size limited by 0.99^n -> 13% yield on 200bp if the process is 99% good
  • Column based / Array based
  • https://www.nature.com/articles/nmeth.2918
• Then assemble them
  • https://en.wikipedia.org/wiki/Gibson_assembly
  • 20-40 bp overlap
  • 5-15 oligos can be combined at once (~2 kb produced)
• New technologies
  • https://static1.squarespace.com/static/5c981af7ebfc7fc8528d6564/t/5f4048cda662a571341b9a60/1598048462430/Alix+-+MDD+DNA+Writing.pdf