Adinulos Week 3

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Purpose

The scientific purpose of the assignment was to evaluate the way the progression of HIV is related to the viral diversity and divergence of strains using the genetic sequences of the strains.

Definitions

seroconverting (seroconversion): the change of a serologic test from negative to positive, indicating the development of antibodies in response to infection or immunization (Biology Dictionary,2020)

viral load: number of viral particles in a sample of a blood sample, in relation to HIV, the load is a surrogate marker for disease progression. Measured by PCR and bDNA tests. (Biology Dictionary, 2020)

synonymous mutation: alternate name for silent mutation, where a nucleotide substitution does not result in an amino acid substitution in the translation product (Oxford Dictionary of Biochemistry and Molecular Biology, 2008)

nonsynonymous mutation: a nucleotide substitution in a protein-coding gene that results in an amino acid substitution in the translation product (Oxford Dictionary of Biochemistry and Molecular Biology, 2008)

chemostats: a device for the continuous culture of bacteria (and other) cells, where growth occurs in an aerated fermenter vessel and its rate is controlled by the rate of addition of fresh nutrients from a reservoir (Oxford Dictionary of Biochemistry and Molecular Biology, 2008).

envelope protein: any protein (usually a glycoprotein) of the envelope of a virus (Oxford Dictionary of Biochemistry and Microbiology, 2008)

proliferation: the reproduction or multiplication of similar forms, especially of cells and morbid cysts (Biology Online, 2020)

predominated: to be superior in number, strength, influence or authority; to have controlling power or influence; to prevail; to rule; to have the mastery (Biology Online, 2020)

stratified: arranged in the form of layers or strata (Biology Online, 2020)

epitopes: any immunological determinant group of an antigen (Oxford Dictionary of Biochemistry and Molecular Biology, 2008)

Outline

Significance

With more interventions and positive immune responses, the number of HIV molecules may decrease, but the genetic diversity and evolution of the virus may not decrease in conjunction with that. These patterns of genetic diversity of the virus are necessary to understand how HIV is adapting to selection forces.

Limitations

Previous studies did not highlight the actual genetic sequence of the viral variants, a large sample, or did not examine the changes of the virus over a long period of time. The researchers were able to overcome these limitations by examining a larger sample of 15 people frequently in the span of 4 years.

Main Resuls

The main result of the paper is that rapid progressor subjects have different patterns of selection, or which type of virus is selected and mutates, in comparison to subjects that were nonprogressing or moderately progressing. Additionally, as the CDT4 cell count declines, the genetic diversity of the HIV virus also increases.

Methods

The experiment was conducted over a 4 year period with a sample of 15 subjects. Each subject was tested at 6-month intervals and had differing amounts of CDT4 cells. Participants were categorized into rapid progressors, moderate progressors, and nonregressors.

A 258-bp region of the env gene was amplified from the blood cells of each of the participants using Nest PCR.
The amplified sequence was cloned and sequenced in using the Sanger termination method using the primers used earlier.
Limiting dilution single-round PCR was used to screen for the number of viral DNA, and 5 copies of the DNA sequence only showed product at the lowest dilution, so a second round of limiting dilution PCR was run. These still showed more than 125 input copies of viral DNA.
Reverse-transcription-PCR was used to determine the number of viral plasma.
Phylogenetic trees were then constructed using the MEGA computer package.
A correlation analysis was run on the data. Data points were determined by 2 visits per person, within a year.
The sequences for each of the individuals were then compared to the tow presented strains. The differences in the strains were classified as either synonymous or nonsynonymous.
The values of the number of dS or dN and the ratio of dS/dN were averaged over all the strains observed.
Participants 9 and 15 showed unusual diversity in their strains, so the researchers constructed three different phylogenetic trees and determined that their viruses presented as monophyletic.
A regression line was fit to compare divergence/diversity over time.

Results

Figure 1. Depicts the participants in groups based on the progression of their disease. The amount of CD4 T cells was plotted on the left y-axis, while divergence (characterized by the median percent of the nucleotides per clone that diverged from the original sequence that was characterized. Diversity was plotted as the mean of the nucleotide differences between the clones. Results for CD4 T cell count over time was variable among all the participants, with mean ranges from increasing 53 cells per year or decreasing 593 cells per year.

Table 1. Depicts the baseline measurements for each of the participants, including the number of observations, the CD4 counts, the median intravisit nucleotide differences among the clones, the virus copy number and the annual rate of decline. CD4 T values were used to categorize subjects into progressive groups. The slope of change in the intravisit nucleotides per clone per year was the measure for diversity, and slope of divergence and median dS/dS ratios were also found. When compared to the baseline, changes between the rate of CD4 T decline were variable among all subjects. In all three progressor categories diversity and divergence increased. Rapid progressors displayed a significant increase in nucleotide differences compared to nonprogressors, indicating that the diversity of the virus increases as the rate of progression increases. When rapid progressors are compared to moderate progressors, rapid progressors also see a positive difference, but is not quite significant. When examining the slope of divergence, rapid progressors also show significant increase when compared to nonprogressors, and while an increased difference was present when compared to moderate progressors, the data was not significant. The median synonymous and nonsynonymous ratios for rapid progressors and moderate progressors was 0.4, which was significant compared to 1. Nonprogressors showed a median value of 1.6 which did not vary significantly compared to 1.

Figure 2. Depicts the average slopes of diversity and divergence over time with the corresponding standard deviation. As the rate of the progression of the disease increases, the diversity of the virus increases. As the rate of the progression of the disease increases, so does the divergence, or the number of nucleotides that are mutated compared to the baseline also increases.

Figure 3. Depicts the phylogenetic tree of subject 9, who showed high genetic variation at the baseline. Visits are indicated by different colors of the branches of the tree. Strains from visit one present at the bottom of the tree. Strains at visit 2 and 3 present at the bottom of the tree. Strains at visit 4 are localized towards the middle of the tree while strains at visit 5 are present throughout the tree indicating that they evolved from strains from different visits, extending from visit 1, visit 3, and other strains from visit 5. Strains from visit 6 emerge from visit 3 and from visit 5.

Figure 4. Depicts phylogenetic trees of 4 randomly selected individuals. Evolution is not sustained within a single branch. Progression does not follow a single branch but returns to strains closely related to strains from earlier visits.

Discussion

A previous study, McDonald et. al. determined that genetic divergence within rapid progressors did increase. However, they found that intravisit diversity between rapid progressors was acutally lower than the slow progressors. Wolinsky et. al. observed that genetic diversity of subjects with rapidly declining CD4 T counts showed was less than those with slower decline. Nowak, found that as CDT 4 cell declined, the genetic diversity of the virus increased.

Support models

The results of this study supported the Nowak model, also mentioned above, where CDT 4 cells decline and viral diversity increases with the progression of the disease. The Sala et al. model describes that the disease progresses by moving away from the virus that (((predominated))) the visit before, which is consistent with the data shown in the phylogenetic trees. The increase of genetic diversity and divergence depicts the continually changing viral strains.

Implications

This work actively highlights that HIV progression works with genetic diversity and divergence to remain present, which means that host immune systems are most likely unable to get rid of HIV because of the sheer range of different strains present.

Future Directions

Some future directions include looking at less replication-competent viruses and developing a more broadly effective immune response.

Critical Evaluation and Limitations

To critique the paper, I would have liked to have seen more information on the process of the statistics. The methods only mentioned a correlation analysis but did not explain how statistical tests were used. The paper was well written and was relevant to current HIV research, it is important to always keep developing research for HIV because it is always evolving. Their results were consistent with previous research, and provide more evidence to Nowak et. al's research. A negative relationship between CD4-T counts and viral genetic diversity and divergence was found in both studies.

Conclusion

The paper aimed to show that patterns of viral selection differ with disease progression types and that genetic diversity is negatively correlated with fewer CDT 4 cell counts. The researchers wanted to study diversity and divergence with a larger sample and use the genetic sequences of the different strains to see the pattern of emergence.

Acknowledgments

I communicated with my partner Jack Menzagopian over text about 10 times to discuss the nature of our figure and any confusions we had.
I followed the protocol on the Week 3 page for this lab entry.
I used the following online dictionaries for unfamiliar terms: Biology Online Dictionary and Oxford Dictionary of Biochemistry and Molecular Biology.
The paper that was analyzed is Markham et. al - Patterns of HIV-1 evolution in individuals with differing rates of CD4 T cell decline
Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Adinulos (talk) 21:35, 1 February 2020 (PST)

References

Biology-Online. (2020). Seroconversion. Retrieved February 1, 2020 from https://www.biology-online.org/dictionary/Seroconversion

Biology-Online. (2020). Viral Load. Retrieved February 1, 2020 from https://www.biology-online.org/dictionary/viral_load

Biology-Online. (2020). Proliferation. Retrieved February 1, 2020 from https://www.biology-online.org/dictionary/proliferation

Biology-Online. (2020). Predominate. Retrieved February 1, 2020 from https://www.biology-online.org/dictionary/Predominate

Biology-Online. (2020). Stratified. Retrieved February 1, 2020 from https://www.biology-online.org/dictionary/Stratified

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

OpenWetWare. (2020). BIOL368/S20:Week 3. Retrieved February 1, 2020, from https://openwetware.org/wiki/BIOL368/S20:Week_3

Oxford Dictionary of Biochemistry and Molecular Biology. (2008). Epitope. Retrieved from https://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-6383?rskey=1Hh1v8&result=1

Oxford Dictionary of Biochemistry and Molecular Biology. (2008). Synonymous Mutation. Retrieved from https://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-19109?rskey=y36d9k&result=1

Oxford Dictionary of Biochemistry and Molecular Biology. (2008). Nonsynonymous Mutation. Retrieved from https://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-13808?rskey=fQGWYV&result=1

Oxford Dictionary of Biochemistry and Molecular Biology. (2008). Chemostats. Retrieved from https://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-3348?rskey=CSaES5&result=1

Oxford Dictionary of Biochemistry and Molecular Biology. (2008). Envelope Protein. Retrieved from https://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-6294?rskey=fg4Voh&result=1