Kam Taghizadeh Week 11

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Searching the Scientific Literature Part 2: Evaluating Scientific Sources

Methods and Results

  1. Evaluated my assigned article in three areas availability, the journal, and the article metadata.
  2. 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 hold the copyright
  3. How is the article available to you:
    1. 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
    2. Evaluating the source--the journal
      1. Who is the publisher of the journal?
        • Elsevier Inc. is the publisher of the journal.
      2. Is the publisher for-profit or non-profit?
        • It is for-profit
      3. Is the publisher a scientific society (some scientific societies partner with a for-profit publisher, some act as their own non-profit publisher)
        • It is not a scientific society
      4. Does the publisher belong to the Open Access Publishers Association?
        • No it does not belong to the Open Access Publishers Association
      5. What country is the journal published in?
        • It was published in the Netherlands
      6. How long has the journal been in operation? (e.g., browse the archive for the earliest article published)
        • 1880
      7. Are articles in this journal peer-reviewed?
        • Yes
    3. Provide a link to the scientific advisory board/editorial board of the journal.
      2. 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
    4. Evaluating the source--the article
      1. Is the article a review or primary research article?
        • It is a primary article.
      2. On what date was the article submitted?
        • It was submitted on April 29,2020.
      3. On what date was the article accepted?
        • It was accepted on June 26,2020.
      4. Did the article undergo any revisions before acceptance?
        • It did not undergo any revisions before acceptance
      5. When was the article published?
        • It was published on July 3, 2020.
      6. What is the approximate elapsed time between submission and publication?
        • Around two months
      7. What are the institutions with which the authors are affiliated?
        • New Mexico Consortium, Los Alamos, NM 87545, USA
        • Duke Human Vaccine Institute & Department of Surgery, Durham, NC 27710, USA
        • Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, NM 87545, USA
        • La Jolla Institute for Immunology, La Jolla, CA 92037, USA
      8. Have the authors published other articles on this subject? (How will you find this out?)
        • Korber has published some articles regarding other viruses such as HIV. I found this out by searching his name in pub med central.
      9. Is there a conflict of interest for any of the authors?
        • There is no conflict of interest.
      10. 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 good article to evaluate further, as it has many figures that are easy enough to understand. Furthermore, it is a very interesting topic to read about, as it provides information on the most prevalent mutation of covid 19.

Conclusion

I have always known how to google scholar search for various biological articles, however I never knew how to find abstract pages until this exercise. I will definetly use this knowledge for future research papers.

Biological Terms and Definitions

  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).
  2. In vitro:Designating biological processes made to occur experimentally in isolation from the whole organism; literally “in glass,” i.e., in the test tube. Examples: tissue cultures, enzyme-substrate reactions. Contrast with in vivo, ex vivo (Cammack et al 2006).
  3. Stochastic:arrived at by skillful conjecture; e.g. a stochastic model, a stochastic process Cammack et al 2006).
  4. Immunogen:any substance that, when introduced into the body, elicits humoral or cell‐mediated immunity, but not immunological tolerance (Cammack et al 2006).
  5. Glycosylation:Substitution of one or more glycosyl groups into a chemical compound or group (Cammack et al 2006).
  6. 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).
  7. 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).
  8. 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).
  9. Polyclonal:an adjective applied to cells or molecules arising from more than one clone; e.g., an antigenic preparation (even a highly purified one) elicits the synthesis of various immunoglobulin molecules. These antibodies would react specifically with different components of the complex antigen molecule. Thus, the antibody preparation generated by such an antigen would be polyclonal in the sense that it would contain immunoglobulins synthesized by different clones of B lymphocytes(Cammack et al 2006).
  10. 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).

Article Outline

Introduction

  1. There have been three major pathogenic zoonotic outbreaks that have occurred due to betacoronaviruses
    • Middle East respiratory syndrome coronavirus (MERS-CoV)
    • Severe acute respiratory syndrome coronavirus (SARS-CoV)
    • SARS-CoV-2, which causes the severe respiratory disease coronavirus disease 2019 (COVID-19)
  2. Antigenic Drift causes mutational changes in influenza viruses
    • This was seen in SARS-CoV
    • Antigenic shift isseen in cold coronaviruses and SARS-CoV
    • Although there is no evidence of this yet in Covid 19, it could acquire it through prolonged human-human transmission
      • It is imperative to study the evolutionary transitions of the antigenic profile of the virus in order to ensure effective vaccines
  3. SARS-CoV-2 immunogens and testing reagents are based on the Spike protein sequence of the Wuhan reference sequence
    • Due to this, they may not have the same effect on mutated strains of SARS-CoV-2
  4. Single amino acids are being monitored due to their phenotypic relevance
  5. 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
    • A bioinformatics pipeline was developed in order to identify spike amino acid variants across many geographic regions by analyzing GISAID information

Results

  1. The analysis pipeline to track SARS-CoV-2 mutations in the COVID-19 pandemic is based on regular updates from the GISAID SARS-CoV-2 sequence database
    • The website provides visualizations and summary data that allow regional tracking of SARS-CoV-2 mutations over time
      • Hundreds of new SARS-CoV-2 sequences are added to GISAID each day
      • The analysis presented is based on a May 29, 2020
        • When Spike alignment included 28,576 sequences
  2. The overall evolutionary rate for SARS-CoV-2 is very low, so a low threshold is set for a Spike mutation to be deemed “of interest.”
    • all sites in Spike where 0.3% of the sequences differ from the Wuhan reference sequence were tracked
    • These sites were monitored for increasing frequency over time in geographic regions as well as for recurrence in different geographic regions.
  3. The Spike D614G amino acid change is caused by an A-to-G nucleotide mutation at position 23,403 in the Wuhan reference strain
    • Found in early March 2020
      • At that time, the G614 form was rare globally but gaining prominence in Europe
    • This was the only spike that met the threshold criterion
  4. The D614G change is accompanied by three other mutations, most of the time:
    • C-to-T mutation in the 5′ UTR (position 241 relative to the Wuhan reference sequence)
    • Silent C-to-T mutation at position 3,037
    • C-to-T mutation at position 14,408 that results in an amino acid change in RNA-dependent RNA polymerase
  5. These 4 genetically linked mutations are now the globally dominant form.
    • Before March 1, 2020, it was found in 10% of 997 global sequences
    • Between March 1 and March 31, 2020, it was found in 67% of 14,951 sequences
    • Between April 1 and May 18, 2020, it was found in 78% of 12,194 sequences.
  6. The transition from D614 to G614 occurred asynchronously in different regions throughout the world, beginning in Europe, followed by North America and Oceania and then Asia.
  7. 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
  8. Used two statistical approaches to assess the consistency and significance of the D614-to-G614 transition:
    • The first statistical approach is a two-sided Fisher’s exact test which compares the counts in the pre-onset 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 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).
  9. The G614 variant increased in frequency even in regions where D614 was the clearly dominant form of a well-established local epidemic when G614 entered the population.
  10. Two exceptions were found to the pattern of increasing G614 frequency
    • The first is Iceland
    • The second is Santa Clara county
  11. The second statistical approach to evaluating the significance of the D614-to-G614 transition uses the time series data in GISAID more fully.
    • Extracted all regional data from GISAID that had a minimum of 5 sequences representing each of the D614 and the G614 variants and at least 14 days of sampling.
  12. A binomial test indicates that G614 increases are highly significantly enriched (p = 2.98e–09).
    • This was also found in 17 of 19 counties/cites (p = 0.0007).
  13. The earliest examples of sequences carrying parts of the 4-mutation haplotype that characterizes the D614G GISAID G clade were found in China and Germany in late January 2020
  14. Cryoelectron microscopy (cryo-EM) structure indicate that the side chains of D614 and T859 of the neighboring protomer (Figure 4B) form a between-protomer hydrogen bond
    • This brings together a residue from the S1 unit of one protomer and a residue of the S2 unit of the other protomer.
  15. G614 Is Associated with Potentially Higher Viral Loads in COVID-19 Patients but Not with Disease Severity
    • Sequences from 999 individuals presenting with COVID-19 disease at the Sheffield Teaching Hospitals NHS Foundation Trust were available and linked to clinical data
  16. Results of RT-PCR supports this increase in infectivity
  17. No significant association between D614G status and disease severity as measured by hospitalization outcomes

Discussion

  1. Over the course of 1 month, the variant carrying the D614G Spike mutation became the globally dominant form of SARS-CoV-2.
  2. Travelers globally dispersed G614 variants and likely would have introduced and reintroduced G614 variants into different locations.
  3. Still, D614 prevalent epidemics were very well established in many locations when G614 first began to appear.
  4. 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
  5. G614 to be 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
  6. Global tracking data show that the G614 variant in Spike has spread faster than D614.
    • This is interpreted to mean that the virus is likely to be more infectious, a hypothesis consistent with the higher infectivity observed with G614 Spike-pseudotyped viruses observed in vitro and the G614 variant association with higher patient Ct values, indicative of potentially higher in vivo viral loads

Implications

  1. The D614G mutation arose independently in the different regions of the world
  2. The D614G mutation has become more prevalent around the world, and its presence indicates how mutations can effect the development of vaccines and therapeutics
  3. D614G had the highest global frequency out of all mutations

Future Directions

  1. The authors should evaluate other amino acid mutations and their prevalence throughout the world in the next coming months.

Critical Evaluations

  1. There could have been a better explanation of the two sided Fisher's exact test for figure 1, so the reader knows exactly what is going on.
  2. There were a lot of different points within this paper, and it was not very focused.

Figure Analysis

  1. Figure 1:
    • The changes in the global distribution of the relative frequencies of D614 which is in orange and G614 which is in blue prior to march 1st on the left and between March 21st and 30th on the right.
    • Bar charts comparing the fraction of sequences with D614 and with G614 for two time periods separated by a 2-week gap.
    • Running weekly average counts of sampled sequences exhibiting the D614 variant in orange and the G614 variant in blue on different continents between January 12 and May 12, 2020.
  2. Figure 2:
    • Changes in the global distribution of the relative frequencies of D614 and G614 variants in Europe prior to march 1st on the left and between March 21st and 30th on the right.
    • Weekly running counts of G614 illustrating the timing of its spread in Europe
  3. Figure S2:
    • The Increasing Frequency of the D614G Variant over Time in North America
  4. Figure S3:
    • The increasing frequency of D614G variant in Australia over time
    • The weekly running frequency of the D614G variant
    • The distribution of D614G variant in Asia
    • The weekly running frequencies of the D614G variant in Asia

Conclusion

This journal article has enlightened me on the fact that the covid 19 strain has already mutated into a form that is becoming more prevalent throughout the world. To know the exact point mutation that has occurred is extremely important, because not many people know the extent to which a viral strain can mutant, or how it even happens. To know this kind of information is very valuable, as I can now say that I have a really good grasp on the nature of this virus. Furthermore, this article has provided a lot of evidence for the increasing frequency of this mutation, and provides very good visuals that the general public can understand as well. Overall, I have learned a lot from this project, and I look forward to learning about any other mutations that could possibly occur.

Powerpoint

File:Journal Club week 11-Korber et al.pdf

Acknowledgements

  • I worked with my partners Owen Dailey, Nathan R. Beshai, and Ian R. Wright to complete our powerpoint
  • 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 powerpoint.
  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Kam Taghizadeh (talk) 01:59, 19 November 2020 (PST)

References

  • 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.
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