Saturday, June 16, 2007

My HIV Research

(note: Im still not online at home-- just stopped in the library to upload this post-- be back soon)


Outside of posting cryptic pictures, or barking at people who intrude upon my territory, I dont talk about my research much here.

But Im about to jump into some heavy duty experiments in the next few weeks, and my posting might drop off a bit, so I figure now is a good opportunity to talk a little bit about what I research.

I study the evolution of HIV over the course of time in HIV+ patients, and how changes to the HIV genome effect fitness (replicative capacity).

Unfortunately, 'fitness' of HIV within a patient depends on a LOT of dimensions. If I dont narrow down what Im studying, I could spend the rest of my life studying the evolution of HIV within one patient. So the first thing I have to do is focus on one region of HIV.

Im interested in the gene 'env'. Env codes for the proteins on the outside of the virus that attach to your cells to infect you. Even more specifically, Im interested in a tiny region (500 base pairs) of env that is very important in HIV tropism (what kind of co-receptor it needs to infect).

Heres what Ive been doing the past couple of years:

Isolate viruses from infected patients over the course of their infection-- 3 months post infection, 6 months, 12 months, etc etc etc.

Amplify the region of the genome that Im interested in with PCR.

Cut and paste the region Im interested in over a common background-- the 'white mouse' of HIV called NL4-3. Just think of it as 'laboratory' HIV. The half lab-half patient HIV viruses are called 'chimeras.'

Now-- the cool part. I added GFP and dsRED2 genes to the HIV genome. So when MY chimeric viruses infect a cell, it will glow green or red.

When I put my viruses on cells in a petri dish, all the chimeras basically look equally 'fit'. Its like letting a hungry 10 year-old kid loose in a room full of Cheetos-- The viruses are just going to go to town.

Im not just studying evolution, I actually need to use evolution in my experiment-- specifically a principle called 'competitive exclusion.' So instead of letting a 10 year-old eat all the Cheetos she wants, I put TWO hungry kids in a room... with a snack-size bag of Cheetos.


One kid is going to eat.


One kid isnt.*



When viruses are in the body, they are competing with eachother for host cells. If I 'compete' viruses against one another in a petri dish, I can determine which of the viruses is more fit. Then I can go back and study the biochemistry of the more fit viruses, and the genetic sequences of the more fit viruses, and figure out why they are more fit.

But wait-- I need to remember my assumptions! Ive filtered out allllll the dimensions in the HIV fitness landscape except the impact of the 500 bp region Im interested in, and in an ex vivo environment. How do I know if the 'fitness' Im measuring is real-world fitness?

There is a phylogenetic tree of all of the isolated viruses. So, say I find a super fit virus at the 6 month time-point--- If it REALLY is more fit, it will leave more offspring, and I can find similar sequences at the 12 month time point, and 18 month, etc! We can also go back and test these viruses in a different cell type, with HIV antibodies present in the competitions, with drugs in the competitions, etc to test different dimentions.


Lots of avenues for further research :)


So, anyway, for the next few weeks I will be spending 8 hours a day doing flow cytometry and real-time PCR, so I wont exactly be in the mood to sit at a computer MORE to write posts... buuuuuut we all know a Creationist or a Denier will say something stupid and Ill get pissed off enough to fight through the inevitable computer overload Ill get at work.


And of course, this is all assuming I even have internet access at home.


*(assume the kids are selfish brats and wont share)

10 comments:

_Arthur said...

"Isolate viruses from infected patients over the course of their infection-- 3 months post infection, 6 months, 12 months, etc etc etc"

So, many newly diagnosed among your patients can pinpoint the precise event of their infection (unfortunately) ?

ERV said...

All of the patients Im studying are infants and children... yeah... Usually they are infected during birth through the eyes/tonsils, so we know exactly when they became infected.

One of the kids Im following was infected in the womb (usually a very bad sign). He is about 10 years old now and doing okay, from what I can gather-- no drugs. Itd be real nice to see how HIV is evolving in this kid, since most infants born with HIV are dead by 3 months.

Ian said...

I had exactly the same question that _Arthur did when I read the post - how does she know when they were infected? (I'm sadder to know the answer...HIV in children is especially tragic, and no, it isn't because they are "less at fault" than adults, it's just because they are children.)

I'm really glad I found my way here (from Pharyngula, iirc). While I knew in the back of my head that HIV was especially prone to mutation, I really hadn't integrated that factoid until I started reading this. Very cool stuff - I especially like the ecological angle of the experiment...competitive exclusion belongs to us (community ecologists), but you're more than welcome to borrow it :)

Stay sane over the next few weeks!

Dustin said...

it isn't because they are "less at fault" than adults

It's nice to hear that. The people in my family are the most calloused fundagelical assholes you'll ever meet, and they've said some pretty bad things about HIV patients, and that's usually sparked when it kills someone they knew. That's pretty fucked up.

If you haven't already, some awesome future posts would be the use of evolutionary distance in HIV for rape cases, and HIV immunity (like that one prostitute in Africa who seems to be immune).

ERV said...

Ian-- Yeah, its depressing. And now you see why I get so angry at Deniers. Theyre out there telling women that anti-retrovirals are poison/will make them sterile/whatever, so women in Africa wont take them, so their kids get HIV when its preventable. If a Denier can look at a 2 year old covered head to toe, inside and out with Kaposis Sarcoma lesions... Jesus.

On a brighter note, community ecologists can have competitive exclusion, but my Cheetos analogy is copyrighted :P

And there will be no staying sane. Ive spent the past +4 hours infecting cells. I have the BEST monster-marks on my face from my goggles and face mask lol!

Dustin-- Theyre quite right. Only bad people get HIV. Or when its not bad people, its to teach bad people a lesson. Or for The Greater Good (TM). Or my favorite-- Gawd gives kids HIV so humans have the opportunity to do good (cure AIDS). Im Gawds SIM character.
Theists dont like talking about the Problem of Evil with me.

Ian said...

And now you see why I get so angry at Deniers.
As much as I despise the creationists, I'd hope there was a special place in hell for HIV denialists (if I believed in hell). Even the average faith-healing charlatan sends people to the doctor when something's seriously wrong. But whether it's these people in Texas letting their daughter die of cancer because they want to try mega-doses of vitamins, or the people who use "chelation" therapy on children with autism (up to 60 injections a month, or so I've read)...

[B]ut my Cheetos analogy is copyrighted
No problem, I'll give you credit for it when I steal it (and steal it I will, at some point in time).

amstar said...

I can't get past the Cheetos image. Soooo much snack food. so little time.

Seriously though. Very interesting project. I love the idea of evaluating rates of infection by having the virus introduce G and RFP.

Is the phylogenetic tree based on the same 500 bp fragment you are using to evaluate fitness or is elsewhere in the genome?

ERV said...

Ian-- If you add a little theatre to the Cheetos analogy, it goes over really well in presentations. Its even funnier if your boss just gave an extremely technical description of your experiment with competitive exclusion, and you stand up and say "Picture a big room full of Cheetos..."

Amstar-- I just thank the Baby Jesus for flow cytometry and real-time PCR. As monotonous as those procedures get, theyre infinitely better than sitting at a microscope counting red and green cells!
We use the entire env gene for our phylogenetic analysis. I think env is the most useful gene for HIV phylogenies, as its the most variable so it gives you the best resolution.

Something else cool about my experiment that Behe wont like: The region Im looking at in env is called 'Variable 3 loop'. In HIV Subtype B, this region has the most variation.

In Subtype C (my samples), Variable 3 is the most conserved.

But according to the HIV expert Michael F. Behe, HIV is all 'the same' :P

Chris Noble said...

But according to the HIV expert Michael F. Behe, HIV is all 'the same' :P

Well it's different from SIV so God must have intervened at the point where it crossed over to humans.

Then again if DaveScot is right then HIV is God's tool to bring about the next stage of human evolution.

The Factician said...

Nice post. Thanks.