Prep for Week 4 Journal Club

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  • CD4 T cell: A form of T lymphocyte with CD4 receptor on the cell surface that recognizes antigens of a virus-infected cell. It releases lymphokines upon stimulation of its recognition of antigens of a virus-infected cell. Upon its antigen recognition it is activated, stimulating, in turn, B lymphocytes and killer T lymphocytes during the antibody formation. It’s the target cell of AIDS virus, infecting and eventually killing the lymphocyte by the virus.

  • Cohort: 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.

  • Seroconversion: The change of a serologic test from negative to positive, indicating the development of antibodies in response to infection or immunization.

  • Nonsynonymous Mutation: A missense mutation - point mutation resulting in a codon that codes for a different amino acid, and thus, causes the synthesis of a protein with an altered amino acid sequence during translation. 
  • Monophylectic: Term applied to a group of organisms which includes the most recent common ancestor of all of its members and all of the descendants of that most recent common ancestor. A monophyletic group is called a clade.

  • Seronegative: serologic evidence of the lack of an antibody of a specific type in the serum; diagnostically useful in ruling out Lyme disease, syphilis, human immunodeficiency virus (HIV), serum hepatitis B, and many other viral diseases.

Mosby's Dental Dictionary, 2nd edition. © 2008 Elsevier, Inc.

  • Epitope: A part of a molecule that an antibody will recognize and bind to.

  • Phylogenetic: (1) Of, or pertaining to phylogenesis.

(2) Relating to the race history of a type of organism.

  • Epidemiology: The study of the distribution and determinants of health-related states and events in populations and the control of health problems, the study of epidemic disease.

  • Nested PCR: Two pairs of PCR primers to be used for a single locus

Patterns of HIV-1 Evolution in Individuals With Differing Rates of CD4 T Cell Decline

I. Background

      A.  HIV-1
               1.  Replicates and mutates rapidly
      B.  Environment types
               1.  Stable
                      a.  one strain becomes dominant
               2.  Unstable
                      a.  may have many effects on virus population
                      b.  may be due to an immune response by host
                      c. or could be due to virus itself
       C.  Destabilizing forces
              1.  Powerful and unselective
                       a.  virus diversity decreases
                       b.  what’s left was likely the most abundant strain 
              2.  Specific
                       a.  decrease in amount of viruses
                       b.  but genetic diversity remains the same
                       c.  diversity will then continue to increase
                                 i.  How do they adapt?
       D.  Previous Research
               1.  Used smaller groups of subjects
               2.  Did not specifically study the DNA sequences
       E.  Current
               1.  Will examine 15 subjects throughout the course of 4 years
               2.  Split up into 3 categories
               3.  Results are contrary to what was previously found
                         a.  higher genetic diversity = lower CD4  cell count

II. Methods

      A.  Subjects
              1.  15 subjects from ALIVE study were used
                       a.  They had HIV and were on a drug regimen
                       b.  divided into 3 groups
                               i.  nonprogressors - CD4 T cells above 650
                               ii.  Moderate progressors - 200-650 CD4 T cell count
                               iii.  Rapid progressors - <200 CD4 T cell count
       B.  Sequencing HIV-1 genes
               1.  Nested PCR was used
               2.  Multiple cycles at various temperatures were run before analysis of clones
               3.  Sequences may be found on GenBank
       C.  Phylogenetic Trees
               1.  Program called MEGA computer package was utilized
               2.  Took precautions to avoid biases
               3.  Color coded the visit times
       D.  Correlation Analysis
               1.  % mutation divergence and % genetic diversity were graphed against CD4 t 			
               cell counts
       E.  dS/dN
               1.  Mutations between strains were categorized
               2.  Used something called Jukes-Cantor correction to avoid bias
               3.  Values had an abnormal distribution, so median was used instead of an average
       F.  Subjects 9 & 15
               1.  Had particularly high genetic variation in comparison to other subjects
                       a.  received special attention
                                i.  Thought that they may have more than 1 type of virus
                                       - turned out to be false

II. Results

       A.  There was a large range of patterns among all 15 subjects
       B.  Statistical significance
                                - ANOVA used
              1.  Moderate vs. Rapid progressor
                      a.  not statistically significant
              2.  Rapid vs. nonprogressor
                      a.  significant
              3.  Moderate vs. nonprogressor
                      a.  significant
       C.  All subjects possessed of homogenous virus population except for subjects 9 & 15
       D.  All 3 categories experienced a rise in genetic diversity and divergence
       E.  There was no 1 strain that was more prevalent than the rest

IV. Discussion

       A.  higher genetic diversity and divergence = lower CD4 t cells
       B.  Synonymous mutation rates were similar across the board
       C. Missense mutations were 3x greater in progressor than nonprogressor
       D.  Selections
                1.  Progressors
                       a.  against non-change
                       b.  for amino acid change
                2.  Nonprogressors
                       a.  against amino acid change
                3.  Against missense mutation is favorable
                       a.  missense mutations result in a more narrow immune response

  • What is the main result presented in this paper?

Prior to these studies, it was believed that lower levels of HIV virus variation were associated with a lower CD4 T cell count. However, a more in-depth experiment with a larger group of subjects indicates the opposite. HIV progression may be more rapid in hosts with greater virus variants.

  • What is the importance or significance of this work?

Hopes were to learn about what forces caused the HIV virus to evolve and the manner in which it is able to adapt. Such information can be useful in determining the best way to attack the virus.

  • What were the limitations in previous studies that led them to perform this work?

Previous studies had a very limited about of subjects and they did not analyze the viruses at the DNA sequence level.

  • What were the methods used in the study?

A 285 bp sequence of the HIV genes were amplified using a Nested PCR. After multiple PCR runs, the sequences were cloned. Phylogenetic trees were generated and correlation analyses were run to compare genetic diversity and CD4 T cell counts. Rates of divergence from the beginning were also examined.

  • Briefly state the result shown in each of the figures and tables.

Table 1: Lists subject, visits, CD4 cell counts, nt differences among clones, and other statistics that was used to draw conclusions about the study.

Fig 1: CD4 cell counts varied greatly among 15 subjects although virus diversity and divergence increased in all.

Fig 2: There are clear differences in diversity and divergence which support the categorization of the subjects

Fig 3: Shows how no 1 strain was dominant. Also shows a pattern mentioned in the article. The virus progresses up to a certain point, then comes (later visits) to near where the 1st visit strains were.

Fig 4: examples of how similar pattern from above is observed in the rest of the subjects

  • How do the results of this study compare to the results of previous studies (See Discussion).

One study (McDonald et al.) showed similar results for 3/5 of their subjects. Another study (Wolinsky et al.) found the opposite to be true. The two subjects studied were both rapid progressors, but possessed viruses with lower genetic diversity.