Mking44 Week 3

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Assignments

Individual Journal Entries

Class Journal Entries

Purpose

  • The purpose of this paper was to examine the evolution of the HIV virus to determine how the variants arose and how they can adapt to forces of selection.

Preparation for Journal Club

Ten Biological Terms

  1. seroconversion (n): "The stage in an immune response when antibodies to the infecting agent are first detected in the bloodstream. For example, people infected with HIV typically seroconvert about 4–6 weeks following the initial infection, when antibodies against viral proteins are first produced,"(Oxford Reference).
  2. nonsynonymous mutation (n): "A nucleotide substitution in a protein‐coding gene that results in an amino acid substitution in the translation product. The ratio of nonsynonymous to synonymous substitutions may be used to detect positive Darwinian selection," (Oxford Reference, 2019).
  3. Long-Term Nonprogressors (LTNP) (n): "A small group of people with HIV who do not take antiretroviral therapy (ART) and still maintain CD4 counts in the normal range indefinitely. The CD4 count is the strongest predictor of HIV progression. A CD4 count less than 200 cells/mm3; indicates that a person has AIDS," (National Institutes of Health).
  4. Peripheral Blood Mononuclear Cell (PBMC) (n): "A type of white blood cell that contains one nucleus, such as a lymphocyte or macrophage," (National Institutes of Health).
  5. neutral mutation (n): "a genetic alteration whose phenotypic expression results in no change in the organism's adaptive value or fitness for present environmental conditions," (Oxford Reference).
  6. differential display (n): "A method, based on the polymerase chain reaction, used to identify genes differentially expressed in various tissues or in normal vs pathological states. The messenger RNA from the two samples is reverse transcribed, amplified with rather nonspecific primers, and run on a high-resolution gel. Bands unique to single samples are considered to be differentially expressed and can be used to clone the full-length cDNA,"(Oxford Reference).
  7. epitope (n): "the antigenic determinant on an antigen to which the paratope on an antibody," (Oxford Reference, 2013).
  8. monophyletic (n): "Applied to a group of species that share a common ancestry, being derived from a single interbreeding," (Oxford Reference, 2010).
  9. chemostat (n): "An apparatus in which a bacterial population can be maintained in the exponential phase of growth by regulating the input of a rate-limiting nutrient and the removal of exhausted medium and cells," (Oxford Reference).
  10. mononuclear (n): "(of a cell) having one nucleus," (Oxford Reference, 2006).

Outline

Introduction

  • There are high mutation rates found in the HIV fast-replicating virus, which can lead to adaptations (evolution) in the host.
  • stable host environment possibility:
    • "best fit" virus: take over rapidly and later mutations will be in the minority of the genetic diversity.
    • different viruses from this environment would have basically the same genes, and variants would not be noticed much because of silent mutations.
  • unstable host environment possibility:a variety of effects could affect the gene pool
    • generated by a complex host immune response (in HIV, changes in co receptors)
    • if it is strong, yet random against a wide set of existing mutants, the genetic makeup would be reduced to those in the few surviving variants, which most likely the mutants that were previously the majority
    • if it only targets the majority in a FDS population (fitness of the genotype depends on its frequency in the population), there could be a reduction in the amount of HIV in the blood without a reduction in genetic diversity, since it only targets the most abundant mutant and not the less common ones.
      • as the diverse variants continue to mutate rapidly, it could lead rise to variants that be greater than the immune response.
  • Therefore, studying the diversity patterns of HIV evolution could give a better understanding into the kind and effectiveness of selection forces that allowed HIV to mutate and how they are able to adapt as well.
  • Previous studies examined a small amount of individuals with HIV, analyzed the evolution using techniques that did not examine direct sequence patterns, and looked at a small amount of time points in each person.
  • Current work examines HIV in 15 subjects from seroconversion (few weeks after the infection, when antibioties are detected in the blood and HIV negative becomes positive) and frequent periods after that up to 4 years.
    • It is better because they can observe people who are non-progressors (virus isn't getting any worse) and people who are rapidly progressing and they found out that there is a different in genetic diversity of the virus.
    • Despite previous work, higher genetic diversity is in rapid CD4 T cell reduction (rapid progressing)

Methods

The Study Population
  • 15 people, 6 month intervals, blood was obtained each visit
  • Rapid progressors: have less than 200 CD4 cells within 2 years
  • Moderate progressors: decline of 200-650 CD4 cells within 4 years
  • Nonprogressors: maintained CD4 T cell levels above 650 through observation period
Sequencing of HIV-1 env Genes
  • Nested PCR, 285 b.p. regio of env gene from PBMC
  • Previous studies shown that the majority of viral DNA is from recently infected PBMC
    • unstable, not integrated into host genome yet, only persists for a few days
    • should be closely related to the RNA in the virus in the blood plasma
  • used two nested primers and two env primers
  • PCR products cloned into plasmid pUC19 and replicated
Plasma Viral Load
  • reverse transcription-PCR
Generation of Phylo-Genetic Trees
  • MEGA computer package, Tamura-Nei distance (corrects base composition and transition/transversion)
  • order of visits in colors: red, orange, green, light blue, dark blue, purple, brown, gray, and black
Correlation Analysis
  • units of analysis= pairs of visits
  • X0=value of diversity(pi) or % mutational divergence
  • Y0=value of CD4 T cells at the visit when X0 determined
  • Y1= value 1 year later
Determination of dS/dN Ratios
  • differences were determined to be synonymous or non synonymous (disregarded random mutations so it would be non bias)
  • irregular distribution, so the median was used.
Examination of Source of Greater Initial Visit Diversity in Subjects 9 and 15.
  • high genetic variation in subject 9 and 15 --> could be infected with two diff viruses.
  • did a phylogenetic tree to see if they were independent or monophyletic
Comparison of the Rate of Change of Divergence and Diversity.
  • fit data with a line, divergence/diversity over time, slope is βi, for the ith subject

Results

  • Two parameters: diversity(average # of nucleotides differences between two clones of the visit), divergence(average % of nucleotides per clone which differed from consensus env sequence)
  • Figure 1: CD4 path, divergence, and diversity over time. circles are cell counts. 200 and 650 are marked with a line. diversity is marked with diamonds, divergence is marked with squares.
    • patterns were variable
  • Table 1: changes of CD4 cells, diversity, and nucleotide divergence in a table for each subject. slopes were variable for each subject.
    • diversity: ranged from -3 to 5 nt per clone, divergence: 0.13% to 2.09% of nucleotides
    • viruses from initial visits of 13/15 subjects were homogeneous (9 and 15 hetero)
  • Figure 2: increase in diversity or divergence was greatest in rapid progressing.
    • significant higher rate of increase in divergence over time and diversity over time than the nonprogressor group (no significant difference than moderate)
    • Both diversity and divergence were significantly negatively correlated with the CD4 cell count a year later
      • subjects who had virus with greater diversity or divergence were more likely to have a greater reduction of CD4 cells
    • ds/dn ratios should be 1 if random.(0.01 for rapid, 0.001 for moderate, 1.6 for nonprogressor)
      • evolution in the nonprogressors does not demonstrate a selective advantage
  • Figure 3: Phylogenetic tree from subject 9, single mutation towards bottom of tree.
    • no evidence of predominance of a single strain throughout the observed time.
  • Figure 4: limited progression pattern shown in randomly selected patients, host factors are not effective for all range of viruses

Discussion

  • higher levels of genetic diversity and divergence in the HIV-1 variants present in a subject had a greater decline in CD4 T cells.
  • nonsynonymous substitutions were found three times more in progressors than nonprogressors
  • strains from nonprogressors showed possible selection against amino acid change, while those from progressors showed selection for such change
  • conflicting evidence in different studies (probably due to looking at seroconversion as a reference)
  • two models to explain:
    • viral clones developing critical epitopes that are outside the T cell repertoire of the host, resulting in failure of the host immune response to control HIV-1 infection.
    • virus observed in the PBMC pool would reflect at which site the cellular environment has become most beneficial to the growth and spread of virus.

Future Research

  • The authors should maybe look at the same subjects for more of an extended period of time, to determine if there are any long-term nonprogressors. Long-term nonprogressors is defined differently in several source, but they have a stable amount of CD4 cells (500-600), for at least 8-10 years after their infection. (Kumar, 2013).
  • The authors should research more into epitopes (if possible) of the HIV virus and determine how they can counteract a host response.

Critiques

  • The authors were very clear on why they wanted to carry out this experiment and the different circumstances of the evolution of the virus. They also compared their experiment to previous experiments to set the grounds on why their experiment was important to finding more data and the limitations of previous experiments. By doing this, it was easier to understand why their experiment was important to read and carry out. They provided the readers with specific methods so they could carry out the same experiment, and the logic behind each methods. They provided their data in organized tables and phylogenetic trees so one could better visualize the findings. They also did well in describing their results and how it supports their conclusion. They did not provide further research one could carry out, but I assume it is not as simple as just performing more experiments. In conclusion, they wrote a great paper, even for someone who is not in the field could read it and understand what their objectives were and how they carried them out.

Scientific Conclusion

  • The HIV virus is known to have a fast mutation rate, as well as, contain variable sequences in its env gene. This gene codes for the proteins that make viruses to infect CD4 T cells. Studying the diversity patterns in HIV could allow for scientists to understand how the virus mutates and how it adapts to the host environment. By examining several HIV patients for an extended period of time, from the point of antibodies being detected, and CD4 cells decreasing, they can examine these patterns. They look at the direct sequences of the virus over time and see how they change, as well as compare the CD4 cell count over time. They found out that higher genetic diversity and genetic divergence were in patients who are rapidly progressing and losing CD4 cells. Their results also show that there is no predominance of a single strain throughout the observed period of time (4 years) and that missense mutations were 3 times more in progressors than nonprogressors. There are two possible models to explain this (1)viruses develop epitopes outside the T cells and (2)viruses observed reflect the site that is most beneficial to virus growth ('hot spots'). Further research is needed to determine which model (if possible) is correct in order to better understand the evolution of HIV viruses

Acknowledgements

  • I worked with my homework partner Maya on APA formatting and determining which biological dictionaries to use
  • I copied and modified the protocol for Week 3 for this assignment
    • Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Mking44 (talk) 14:20, 30 January 2020 (PST)

References

  1. (2006). mononuclear. In 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 2 Feb. 2020, from https://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-12889.
  2. (2019). monophyletic. In Allaby, M. (Ed.), A Dictionary of Plant Sciences. : Oxford University Press. Retrieved 2 Feb. 2020, from https://www.oxfordreference.com/view/10.1093/acref/9780198833338.001.0001/acref-9780198833338-e-4339.
  3. (2019). seroconversion. In Hine, R. (Ed.), A Dictionary of Biology. : Oxford University Press. Retrieved 30 Jan. 2020, from https://www.oxfordreference.com/view/10.1093/acref/9780198821489.001.0001/acref-9780198821489-e-6465
  4. differential display PCR. Oxford Reference. Retrieved 30 Jan. 2020, from https://www.oxfordreference.com/view/10.1093/oi/authority.20110803095717744.
  5. King, R., Mulligan, P., & Stansfield, W. (2013). epitope. In A Dictionary of Genetics. : Oxford University Press. Retrieved 2 Feb. 2020, from https://www.oxfordreference.com/view/10.1093/acref/9780199766444.001.0001/acref-9780199766444-e-2141.
  6. Kumar, P. (2013). Long term non-progressor (LTNP) HIV infection. The Indian journal of medical research, 138(3), 291.
  7. Lackie, J. chemostat. In Nation, B. (Ed.), A Dictionary of Biomedicine. : Oxford University Press. Retrieved 2 Feb. 2020, from https://www.oxfordreference.com/view/10.1093/acref/9780191829116.001.0001/acref-9780191829116-e-1709.
  8. "Long-Term Nonprogressors (LTNP) Definition.” National Institutes of Health, U.S. Department of Health and Human Services, 30 Jan. 2020, aidsinfo.nih.gov/understanding-hiv-aids/glossary/423/long-term-nonprogressors.
  9. Markham, R. B., Wang, W. C., Weisstein, A. E., Wang, Z., Munoz, A., Templeton, A., ... & Yu, X. F. (1998). Patterns of HIV-1 evolution in individuals with differing rates of CD4 T cell decline. Proceedings of the National Academy of Sciences, 95(21), 12568-12573. doi: 10.1073/pnas.95.21.12568
  10. nonsynonymous mutation. Oxford Reference. Retrieved 30 Jan. 2020, from https://www.oxfordreference.com/view/10.1093/oi/authority.20110803100238383.
  11. neutral mutation. Oxford Reference. Retrieved 30 Jan. 2020, from https://www.oxfordreference.com/view/10.1093/oi/authority.20110803100230492.
  12. “Peripheral Blood Mononuclear Cell (PBMC) Definition.” National Institutes of Health, U.S. Department of Health and Human Services, 30 Jan. 2020, aidsinfo.nih.gov/understanding-hiv-aids/glossary/818/peripheral-blood-mononuclear-cell.
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