Mpaniag1 Week 3

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The purpose of this research was to examine the patterns of HIV and how selection forces can influence evolution, but most importantly how the virus is able to mutate.

10 Biological Terms

  1. Seroconverting-v. to produce specific antibodies in response to the presence of an antigen e.g. a vaccine or a virus. ("Seroconvert: Meaning of Seroconvert by Lexico")
  2. T cell-A type of white blood cell. T cells are part of the immune system and develop from stem cells in the bone marrow. They help protect the body from infection and may help fight cancer. Also called T lymphocyte and thymocyte. (National Cancer Institute)
  3. Monophyletic-A taxonomic group that holds characters descended from a single common ancestor. (Biology Online Dictionary)
  4. Neutral Mutations-A mutation that has no selective advantage or disadvantage. Considerable controversy surrounds the question of whether such mutation exists. (The Dictionary of Cell and Molecular Biology, 2007, p. 288)
  5. Peripheral Blood mononuclear cells-A mixture of monocytes and lymphocytes; blood leucocytes from which granulocytes have been separated and removed. (The Dictionary of Cell and Molecular Biology, 2007, p. 317)
  6. Proliferation- The reproduction or multiplication of similar forms, especially of cells and morbid cysts. (Biology Online Dictionary)
  7. Cohort-a cohort is a group of animals of the same species, identified by a common characteristic, which are studied over a period of time as part of a scientific or medical investigation. (Biology Online Dictionary)
  8. Divergence-(evolutionary biology) The evolutionary process wherein a population of species diverge into two or more descendant species, resulting in once similar or related species becoming more and more dissimilar (Biology Online Dictionary)
  9. Epitope-that part of an anti-genic molecule against which a particular immune response is directed-or instance, a tetra- to pentapeptide sequence in a protein, a tri- to a penta-glycoside sequence in a polysaccharide (The Dictionary of Cell and Molecular Biology, 2007, p. 30)
  10. Hypervariable region-those regions of the heavy or light chains of immunoglobulins in which there is considerable sequence diversity within that set of immunoglobins in a single individual. These regions specify the antigen affinity of each antibody. (The Dictionary of Cell and Molecular Biology, 2007, p. 209)



  • HIV-1 env sequences were studied in users with different CD4 T cell decrease
  • The pattern suggests either selection against the main virus or the virus evolving independently
  • Different CD4 T cell levels show different levels and types of mutations


  • Because HIV has high rates of mutation and replication it is able to combat changes that occur in its host
  • Changes in the host can causes the genetic composition of the virus to change
  • A destabilizing force would not discriminate against the different variants, resulting in less genetic diversity
  • A selective force would select for the most numerous variant, genetic diversity would not be lost
  • Mutation would increase the genetic diversity
  • If there was so much genetic diversity and not one variant was prominent than selective forces would not work as well
  • Therefore researching the diversity of HIV can provide information about the selective forces mechanisms and regulation and how to virus combats against those forces

Previous Studies

  • Studied very small groups
  • Used techniques that did not analyze sequences directly
  • Limited number of points

Current Study

  • 15 subjects, seroconversion-period of four years

What is shown?

  • Nonprogressors have different selection methods than rapid progressors
  • High genetic diversity=CD4 T cell levels decrease


The Study Population

  • 15 subjects pooled from the ALIVE study in Baltimore, MD
  • Collected blood samples from each of the subjects every six months, those blood samples were then analyzed
  • What the different levels of CD4 cells mean?
    • Rapid progressors-200 CD4 T cells
    • Moderate-200-650 CD4 T cells
    • Nonprogressors-above 650 CD4 T cells

Sequencing of HIV-env Genes

  • Nested PCR-285 bp region of env gene
  • Env gene obtained from peripheral blood mononuclear cells
  • High viral DNA levels in recently infected but not yet activated peripheral blood mononuclear cells
  • Used two primers and two nested primers
  • BamHI and EcoRI sites were included in the nested primers
  • PCR Cycle
  1. 2 min at 95 degrees
  2. 30 secs at 94 degrees
  3. 30 secs at 60 degrees
  4. 45 secs at 72 degrees
  5. repeat steps 2-5 35 times
  6. 10 min at 72 degrees
  7. store at 4 degrees
  • PCR DNA was cloned into pUC19
  • DNA was sequenced using Sanger Sequencing

Plasma Viral Load

  • Used reverse transcription PCR

Generation of Phylogenetic Tree

  • Trees were made using MEGA
  • Taxa were colored based off of the time observed
    • Red-V1
    • Orange-V2
    • Green-V3
    • Light Blue-V4
    • Dark Blue-V5
    • Purple-V6
    • Brown-V7
    • Gray-V8
    • Black-V9
  • A phylogenetic tree showed when the clones segregated from one another

Correlation Analysis

  • X0=percent mutational divergence
  • Y0=CD4 T cell count at X0
  • Y1=CD4 T cell count a year later

Determination of dS/dN ratios

  • Each previous sample was compared to the present-i.e. sample 1 with sample 2, sample 2 with sample 3, etc
  • dS, dN, and their ratio were averaged to eliminate bias

Examination of Source of greater Initial Visit Diversity in Subjects 9 and 15

  • Subjects 9 and 15 demonstrated high levels of genetic diversity in first sample
  • Analyzed as independent or monophyletic virus

Comparison of the Rate of Change of Divergence and Diversity

  • Each subject has a graph (divergenence/diversity over time) fit with a line of regression
  • Find slope
  • Take averages of the slopes of progressors, moderate progressors, and nonprogressors


  • Fig 1-CD4 T cell diversity over a certain period of time, the pattern of decline was quite different among the subjects
  • Table 1-Data taken from seroconverters, range varied year to year i.e. 1 subject increased 53 CD4 T cells, 1 subject lost 593 CD4 T cells
  • Used V3 region because able to handle mutations
  • Genetic Diversity and Divergence were observed from sample to sample, and visit to visit
  • Both increased over time
  • Fig 2-Divergence and Diversity were greater in rapid Progressors
  • Fig 2A-Diversity was greater in rapid progressors
  • Fig 2B-Divergence was greater in rapid progressors
  • Difference between progressors and moderate progressors was not statistically significant
  • Diversity and Divergence were negatively correlated with CD4 T cells
  • dS/dN ratio for rapid and moderate progressors was 0.4, non-synonymous mutations favored
  • Fig 3-Phylogenetic trees showed no single strain dominated throughout the experiment
  • Fig 4-Evolution is not maintained from only 1 branch


  • Higher genetic diversity and divergence=Decline in T cells (CD4)
  • Synonymous mutations about the same between the three groups
  • Non synonymous mutations, progressors 3 times as much than in nonprogressors

Previous Studies

  • Found that diversity in rapid progressors was less than slow progressors-McDonald et al/Wolinsky et al


  • Model 1-increasing diversity, followed by T cell decline, epitopes form and the T cell, fails to get an immune response from the host
  • Model 2-cell environment is constantly changing because of T cell activation or immune response


In conclusion, this paper did explore the patterns of HIV and found that rapid progressors have higher rates of diversity and divergence than moderate or non-progressors. Rapid and moderate progressors evolved favoring the nonsynonymous mutation while nonprogressors selected against it.


The implications of this paper are that they learn more about the virus, HIV, leading to the ability to find a way to suppress the disease of AIDS from progressing.

Future Directions

The author can study the role of epitopes in diversity. Another direction the author can take is to use a different region of the env, to see if the results are the same.


The other did a very good job of explaining each step of the process. I understood how they carried out their experiment, the results that came from it, and the discussion. They backed up their data when other studies found something different. However, I wish they would have gone deeper into the applications and future research that can take place as a result of their findings.


  • I worked and discussed with Madeleine B. King on this homework assignment, she helped me find words that I could define and explained information to me
  • I emailed Kam D. Dahlquist, Ph.D. one question to clarify how to the references for a source that has been used multiple times
  • I copied the citation for the paper for the Week 3 assignment page
  • I copied and modified wiki syntax for how to bold from Week 3 assignment page <Kam D. Dahlquist>
  • I copied and modified the protocol for Week 3 assignment
  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Mpaniag1 (talk) 10:18, 4 February 2020 (PST)





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