Owen R. Dailey Week 11
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Assignments
Individual Journals
The D614G Research Group Week 12
The D614G Research Group Week 14
Class Journals
Purpose
The purpose of this assignment is to find the effect of the D614G mutation on the SARS-CoV-2 virus
Methods/Results
Evaluating the Scientific Literature
- Now we will begin to evaluate your assigned article in three areas availability, the journal, and the article metadata. Again, provide a citation for the article in APA format, this time including the DOI. For the following questions, for information that is not available, answer n/a).
- Korber B, Fischer WM, Gnanakaran S, Yoon H, Theiler J, Abfalterer W, Hengartner N, Giorgi EE, Bhattacharya T, Foley B, Hastie KM, Parker MD, Partridge DG, Evans CM, Freeman TM, de Silva TI; Sheffield COVID-19 Genomics Group, McDanal C, Perez LG, Tang H, Moon-Walker A, Whelan SP, LaBranche CC, Saphire EO, Montefiori DC. Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus. Cell. 2020 Aug 20;182(4):812-827.e19. doi: 10.1016/j.cell.2020.06.043. Epub 2020 Jul 3. PMID: 32697968; PMCID: PMC7332439. DOI: 10.1016/j.cell.2020.06.043.
- Provide a link to the abstract of the article on PubMed
- Provide a link to the full text of the article in PubMed Central
- Provide a link to the full text of the article (HTML format) from the publisher website.
- Provide a link to the full PDF version of the article from the publisher website.
- Who owns the rights to the article? Look at the first page of the PDF version of the article for the © symbol. Generally, either the journal/publisher or the authors will hold the copyright.
- The authors own the right to the article
- How is the article available to you:
- Is the article available “open access” (look for the words “open access” or the “unlocked” icon on the article website or the first page of the PDF) If YES, stop here.
- Yes
- Is the article available “open access” (look for the words “open access” or the “unlocked” icon on the article website or the first page of the PDF) If YES, stop here.
- Evaluating the source--the journal
- Who is the publisher of the journal?
- Elsevier Inc.
- Is the publisher for-profit or non-profit?
- For-profit
- Is the publisher a scientific society (some scientific societies partner with a for-profit publisher, some act as their own non-profit publisher)
- Not a scientific society
- Does the publisher belong to the Open Access Publishers Association?
- No
- What country is the journal published in?
- The Netherlands
- How long has the journal been in operation? (e.g., browse the archive for the earliest article published)
- 1880
- Are articles in this journal peer-reviewed?
- Yes
- Provide a link to the scientific advisory board/editorial board of the journal.
- What is the journal impact factor (look to see if it is provided on the journal home page; often you can also find it through a Google search)?
- 3.481
- Who is the publisher of the journal?
- Evaluating the source--the article
- Is the article a review or primary research article?
- Primary
- On what date was the article submitted?
- April 29, 2020
- On what date was the article accepted?
- June 26, 2020
- Did the article undergo any revisions before acceptance?
- No
- When was the article published?
- July 3, 2020
- What is the approximate elapsed time between submission and publication?
- ~2 months
- What are the institutions with which the authors are affiliated?
- Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, NM 87545, USA
- New Mexico Consortium, Los Alamos, NM 87545, USA
- Duke Human Vaccine Institute & Department of Surgery, Durham, NC 27710, USA
- La Jolla Institute for Immunology, La Jolla, CA 92037, USA
- Have the authors published other articles on this subject? (How will you find this out?)
- Korber has publishd quite a few articles on other viruses, naemly HIV
- Is there a conflict of interest for any of the authors?
- No
- Make a recommendation--based just on the information you have gathered so far, is this a good article to evaluate further? Why or why not?
- This is a goog article to evaluate further. Published in a well respected journal by authors who do not have any bias and who have done research on past viruses
- Is the article a review or primary research article?
- For your Conclusion section of this individual assignment, write a short reflection about what you learned by doing this exercise. Include in your answer what you knew previously about searching the biological literature and evaluating sources, and what you learned that was new that you learned today.
Biological Terms and Definitions
- Titer: "A measure of the concentration or activity of an active substance, e.g. an antibody, in a solution, usually expressed as the highest dilution of the solution in which the activity can be detected. By convention, if the highest dilution giving activity is 100‐fold, the titre is said to be 100" (Cammack et al 2006).
- Oxford Dictionary of Biochemistry and Molecular Biology. (2006). Titre. Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. Oxford University Press. Retrieved November 14, 2020, from https://www-oxfordreference-com.electra.lmu.edu/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-19642?rskey=pZSlBQ&result=2
- Virion: "A complete virus particle, found extracellularly and capable of surviving in crystalline form and of infecting a living cell. It comprises the nucleic‐acid core and the protein capsid; the latter may be enclosed by an envelope in some viral families" (Cammack et al 2006).
- Oxford Dictionary of Biochemistry and Molecular Biology. (2006). Virion. Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. Oxford University Press. Retrieved November 14, 2020, from https://www-oxfordreference-com.electra.lmu.edu/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-20603?rskey=9KBCXm&result=1
- Antigenic Drift: "Any of the minor changes in the antigenicity of influenza virus that result from spontaneous mutation, with corresponding minor changes in the amino‐acid sequence of the viral hemagglutinin" (Cammack et al 2006).
- Oxford Dictionary of Biochemistry and Molecular Biology. (2006). Antigenic Drift. Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. Oxford University Press. Retrieved November 14, 2020, from https://www-oxfordreference-com.electra.lmu.edu/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-1289?rskey=osmO64&result=1
- Immunogen: "Any substance that, when introduced into the body, elicits humoral or cell‐mediated immunity, but not immunological tolerance. Any substance that is able to stimulate an immune response, as distinct from substances (antigens) that can combine with antibody. any substance that is able to stimulate protective immunity against a pathogen, as distinct from an immune response that is of no intrinsic value to the animal immunized" (Cammack et al 2006).
- Oxford Dictionary of Biochemistry and Molecular Biology. (2006). Immunogen. Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. Oxford University Press. Retrieved November 14, 2020, from https://www-oxfordreference-com.electra.lmu.edu/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-9819?rskey=FIhBPQ&result=1
- Haplotype: "A set of genes located on a single chromosome; the term is used also to denote the characteristics dependent on those genes. In outbred populations the maternal and paternal chromosomes usually differ, so an individual has two haplotypes, one derived from each parent" (Cammack et al 2006).
- Oxford Dictionary of Biochemistry and Molecular Biology. (2006). Haplotype. Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. Oxford University Press. Retrieved November 14, 2020, from https://www-oxfordreference-com.electra.lmu.edu/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-8632?rskey=rAfute&result=1
- Stochastic: "Arrived at by skilful conjecture" (Cammack et al 2006).
- Oxford Dictionary of Biochemistry and Molecular Biology. (2006). Stochastic. Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. Oxford University Press. Retrieved November 14, 2020, from https://www-oxfordreference-com.electra.lmu.edu/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-18788?rskey=1AMvGC&result=1
- Glycosylation: "Substitution of one or more glycosyl groups into a chemical compound or group" (Cammack et al 2006).
- Oxford Dictionary of Biochemistry and Molecular Biology. (2006). Glycosylation. Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. Oxford University Press. Retrieved November 14, 2020, from https://www-oxfordreference-com.electra.lmu.edu/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-8256?rskey=aM8DCX&result=1
- Protease: "any enzyme that catalyses the hydrolysis of peptide bonds in a protein or polypeptide" (Cammack et al 2006).
- Oxford Dictionary of Biochemistry and Molecular Biology. (2006). Protease. Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. Oxford University Press. Retrieved November 14, 2020, from https://www-oxfordreference-com.electra.lmu.edu/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-16481?rskey=3Mdeon&result=1
- Real-Time PCR: "A variant of the polymerase chain reaction (PCR) in which the quantity of amplified DNA is recorded in each cycle by monitoring the fluorescence intensity of a DNA‐binding dye such as SYBR Green. An advantage is that the kinetics of the reaction can be monitored and, with reference to the behaviour of standards, the amount of PCR product quantified. The abbreviation RT‐PCR for real‐time PCR is to be deprecated. Real‐time PCR is frequently used for quantitation of reverse transcriptase PCR in which case real‐time RT‐PCR is the appropriate terminology" (Cammack et al 2006).
- Oxford Dictionary of Biochemistry and Molecular Biology. (2006). Real-Time PCR. Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. Oxford University Press. Retrieved November 14, 2020, from https://www-oxfordreference-com.electra.lmu.edu/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-16995?rskey=RWsG1r&result=10
- Infectious: "Capable of being transmitted to another organism" (Cammack et al 2006).
- Oxford Dictionary of Biochemistry and Molecular Biology. (2006). Infectious. Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. Oxford University Press. Retrieved November 14, 2020, from https://www-oxfordreference-com.electra.lmu.edu/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-9939?rskey=7Zg8jl&result=1
Article Outline
Introduction
- Three major pathogenic disease outbreaks caused by betacoronaviruses
- SARS-COV
- MERS-CoV
- SARS-CoV-2
- Coronaviruses have proofreading mechanisms
- Genetic diversity between SARS-CoV-2 sequences is low
- Natural selection still has the ability to produce genetic mutations that are favorable for viral infection
- For example, antigenic drift in the influenza virus occurs due to immunological mutations and the fitness landscape
- The duration of the SARS-Cov-2 pandemic may enable the accumulation of immunologically relevant mutations
- Antigenic shift occurs in the common cold coronaviruses and SARS-Cov-1
- It is important to monitor these gradual mutations and how they effect vaccine and therapeutic effectiveness
- Most current SARS-CoV-2 immunogens and testing reagents are based on the Spike protein sequence of the Wuhan reference sequence
- Thus, they may not have the same effect on SARS-CoV-2 strains that have been mutated
- Phylogenetic analysis of the global sampling of SARS-CoV-2 is being very capably addressed by the Global Initiative for Sharing All Influenza Data (GISAID) database
- Korber et al. developed an alternative indicator of potential positive selection by identifying variants that are recurrently becoming more
prevalent in different geographic locations
- If increases in relative frequency of a particular variant are observed repeatedly in distinct geographic regions, then that variant becomes a candidate for conferring a selective advantage
- Single amino acid changes are worth monitoring because they can be phenotypically relevant
- Can confer resistance to antibodies (MERS-CoV, SARS-CoV-1)
- Alter host species susceptibility (HIV)
- Increase expression levels (HIV)
- Change viral phenotype (HIV)
- Confer complete or nearly complete resistance to classes of neutralizing antibodies (HIV)
Results
- Analyzed all sequences from the GISAID SARS-CoV-2 sequence database
- Analysis updated daily
- Website provides visualizations and summary data that allow regional tracking of SARS-CoV-2 mutations over time
- Analysis in this paper is from the May 29, 2020 update of GISAID SARS-CoV-2 sequence database
- 28,576 spike protein sequences
- Tracked spike mutations where 0.3% of the sequences differ from the Wuhan reference sequence
- Only 0.3% threshold because the evolutionary rate for SARS-CoV-2 is very low
- The paper only analyzes the D614G mutant sequence
- The Spike D614G amino acid change is caused by an A-to-G nucleotide mutation at position 23,403 in the Wuhan reference strain
- D614G mutant strain is usually a haplotype mutation
- D614G change is almost always accompanied by three other mutations: a C-to-T mutation in the 50 UTR (position 241 relative to the Wuhan reference sequence), a silent C-to-T mutation at position 3,037, and a C-to-T mutation at position 14,408 that results in an amino acid change in RNA-dependent RNA polymerase (RdRp P323L)
- The transition from D614 to G614 occurred asynchronously in different regions throughout the world
- To observe a significant change in the frequency of variants in a geographic region, three requirements must be met
- Both variants must at some point be co-circulating in the geographic area
- There must be sampling over an adequate duration to observe a change in frequency
- Enough samples must be available for adequate statistical power to detect a difference
- Two statistical approaches to assess the consistency and significance of the D614-to-G614 transition
- Two-Sided Fisher’s Exact Test: Compares the counts in the preonset period with the counts after the 2-week delay period and provides a p value against the null hypothesis that the fraction of D614 versus G614 sequences did not change
- Almost all places that were analyzed shifted toward increasing G614 frequencies: 5 of 5 continents, 16 of 17 countries (two-sided binomial p value of 0.00027), 16 of 16 regions (p= 0.00003), and 11 of 12 counties and cities (p = 0.0063)
- Binomial Test: Compares the fraction of D614 to G614 over time with the null hypothesis that this fraction does not change over time
- Then separately tested the null against two alternative hypotheses: that the fraction of G614 increases or that it decreases
- 30 of 31 subcountries/states with a significant change in frequency were increasing in G614
- Then separately tested the null against two alternative hypotheses: that the fraction of G614 increases or that it decreases
- Two-Sided Fisher’s Exact Test: Compares the counts in the preonset period with the counts after the 2-week delay period and provides a p value against the null hypothesis that the fraction of D614 versus G614 sequences did not change
- The earliest recorded date of the 4 mutation haplotype were found in China and Germany in late January 2020
- Had 3/4 mutations
- February 20, 2020 is the earliest recorded date of the 4 mutation haplotype with all 4 mutations
- Structural implications of the D614G mutation
- The side chains of D614 and T859 of the neighboring protomer form a between-protomer hydrogen bond, bringing together a residue from the S1 unit of one protomer and a residue of the S2 unit of the other protomer
- The G614 mutation would eliminate this H bond possibly increasing mainchain flexibility and altering between-protomer interactions, and might modulate glycosylation at the nearby N616 site
- The side chains of D614 and T859 of the neighboring protomer form a between-protomer hydrogen bond, bringing together a residue from the S1 unit of one protomer and a residue of the S2 unit of the other protomer
- G614 Is Associated with Potentially Higher Viral Loads in COVID-19 Patients but Not with Disease Severity
- Results of RT-PCR supports this increase in infectivity
- Increase in infectivity does not increase mortality however
- No significant association between D614G status and disease severity as measured by hospitalization outcomes
- G614 Is Associated with Higher Infectious Titers of Spike-Pseudotyped Virus
- Additionally, despite increased fitness in cell culture, G614-bearing virions are not intrinsically more resistant to neutralization by convalescent sera
Discussion
- Over the course of 1 month, the variant carrying the D614G Spike mutation became the globally dominant form of SARS-CoV-2
- The mutation that causes the D614G amino change is transmitted as part of a conserved haplotype defined by 4 mutations that almost always track together
- G614 is associated with higher levels of viral nucleic acid in the upper respiratory tract in human patients, suggestive of higher viral loads, and with higher infectivity in multiple pseudotyping assays
- Global tracking data show that the G614 variant in Spike has spread faster than D614
- G614 is more infectious
- No evidence that G614 confers higher severity
- It will be important to determine whether the D614 and G614 forms of SARS-CoV-2 are differentially sensitive to neutralization by vaccine-elicited antibodies or by antibodies produced in response to infection with either form of the virus
- Limitations of the study
- Shifts in frequency toward the G614 variant be because founder effects or sampling biases
- Infectiousness and transmissibility are not always synonymous, and more studies are needed to determine whether the D614G mutation actually led to an increase in number of infections and not just higher viral loads during infection
Implications
- Mutations in the SARS-CoV-2 Genetic Sequence Can Alter the Effectiveness of Vaccines and Therapeutics
- There were Multiple Spike Sites and Mutations of Interest, but the D614G Mutation had the Highest Global Frequency
- The D614G Mutation Arose Independently in Different Regions of the World
- G614 Mutants Grow to a Higher Concentration in Cell Cultures than their D614 Counterparts
Future Directions
- See if the G614 mutants grow to higher concentration in lung cells than D614
- Test to see if antibodies generated from patients with the D614 form can neutralize the G614 form
Critical Evaluations
- In general, the paper supports its conclusions very well
- The parsimony trees are very complex given that there 17,000 sequences and I'm not sure if they are very easy to read and that helpful to the paper
Figure Analysis
- Figure 6
- G614 Mutants Grow to a Higher Concentration in Cell Cultures than their D614 Counterparts
- Despite Being More Infectious, G614 Mutants are Not More Resistant to Polyclonal Antibodies than their D614 Counterparts
- The convalescent serum was collected from six people in San Diego.
- It is uncertain what form of the virus they were infected with.
- “G614 is associated with potentially higher viral loads in COVID-19 patients but not with disease severity” (Korber et al., 2020)
- Figure 7
- There were Multiple Spike Sites and Mutations of Interest, but the D614G Mutation had the Highest Global Frequency
- 17,760 D614G mutations were found in the GISAID database by June 2, 2020.
- Many mutations were contained to one geographic region, but D614G was a global mutation
- There were Multiple Spike Sites and Mutations of Interest, but the D614G Mutation had the Highest Global Frequency
- Figure S6
- There are a large number of sequences from Europe.
- The Order of the D614 to G614 Transition: Europe -> North America -> Oceania -> Asia
Conclusion
Acknowledgements
- I worked with my Kam Taghizadeh, Nathan R. Beshai, and Ian R. Wright to complete our journal club presentation
- I copied and modified the procedures shown on the Week 11 page.
- I used the Korber et al. paper for the journal club outline and presentation.
- Except for what is noted above, this individual journal entry was completed by me and not copied from another source.
Owen R. Dailey (talk) 16:59, 18 November 2020 (PST)
References
- Specific reference for each biological definition can be found under it in the biological term section
- Korber, B., Fischer, W. M., Gnanakaran, S., Yoon, H., Theiler, J., Abfalterer, W., ... & Hastie, K. M. (2020). Tracking changes in SARS-CoV-2 Spike: evidence that D614G increases infectivity of the COVID-19 virus. Cell, 182(4), 812-827.
