Author Topic: This is what poses for scientific thinking amoung certain retrovirologists today  (Read 5125 times)

omerbasket

  • Hero Member
  • *****
  • Posts: 575

Could you double-check that?  How can it be 100% identical to gag sequences from those different viruses if these are BLAST analyses for the whole gag gene? (Are they?)  The Lo/Alter fragments were missing the gag leader deletion, so they can't be 100% similar over all of gag to viruses that have the deletion.  There also must be at least a couple nucleotides off here and there.. And all those other viruses are not 100% similar to each other, so again there can't be 100% identity with all of them.  Could you please re-check that?  Perhaps I am interpreting something wrong, but that BLAST result doesn't sound right on the face of it.
First of all - I have to say that I don't understand well the scientific bases here. That is why we need scientists to interpret this.

Anyway, I did check it again and the results were the same. I took a printscreen of that, so that you can look. I can't printscreen the title of the BLAST results and the comparisons to known sequences - but since the BLAST tool also writes the same sequence that was looked at in the comparison results (for example, if you look for "what is similar to VP62", you would also find in your results "VP62"), you can see that it is the "MLV-related virus CFS isolate MLV001-2010 gag polyprotein (gag) gene, partial cds" that I'm looking at. You can see that if you look at the blue box and at the small red box that shows that it has the same accession number (I think that every sequence has it's own very unique accession number). I also used the large red box in order to show you that it's 100% similar to VP62, VP42 etc (scroll to the right in order to see the rest of the picture below).


That being said, when I've scrolled down to look at the actual nucleotides - I saw that in everyone of these sequences there is one nucleotide change compared to MLV-001 - while MLV-001 have a G in it's 71st nucleotide, the other viruses (VP62, VP42, VP35, the WPi's ones and the 22Rv1 one) has an A in the compared location (it's nucletoide number 705 in them - that is because they are fully sequences, and MLV-001 is only partially sequenced). The reason the "BLAST" tool said it's 100% identical is that there are 276 identical nucleotides out of 277, which means it is 99.639 identical, so it rounded that up to 100%. I checked another sequence for which the "BLAST" said was 99% identical, and it was about 99.2% identical. So I guess that anything from 99.5% and above it counts as 100%. Anyway, You can see it for yourselves:



Scientists, we really need your help. in order to check for the viruses found by the Lo/Alter study what you need to do is to go to www.ncbi.nlm.nih.gov, run a search with the word "CFS", and then click on "nucleotide" (it's in the fourth line on the right side), There you will have the 14 sequences found in the Lo/Alter study.For each sequence, in order to compare it to similar known sequences, you should click on the link of the sequence, in the next page click "Run BLAST" on the right side, and then click "BLAST", below on the left side. Then you will have the BLAST results. What's more important that confirming that you see what I see - is to confirm if the conclusions are correct, given that we only have parital sequences from the study of Lo/Alter.
« Last Edit: March 09, 2011, 08:55:08 PM by omerbasket »

omerbasket

  • Hero Member
  • *****
  • Posts: 575
"Dr. Yes", as you requested, I've checked for the results of comparing the published WPI sequences to the proviral XMRV from 22Rv1 full genome. These are the results:

1) "Xenotropic MuLV-related virus isolate WPI-1169 putative polyprotein gene, partial cds" (accession: GQ483510.1) - it is 98% identical to the proviral XMRV from 22Rv1 full genome, with a 99% of query coverage.

2) "Xenotropic MuLV-related virus isolate WPI-1138 putative polyprotein gene, partial cds" (accession: GQ483509.1) - it is 99% identical to the 22Rv1 XMRV sequence, with a 99% of query coverage.

3) "Xenotropic MuLV-related virus isolate WPI-1130 putative polyprotein gene, partial cds" (accession: GQ483508.1) - it is 99% identical to the 22Rv1 XMRV sequence, with a 99% of query coverage.

4) "Xenotropic MuLV-related virus clone WPI-1104 putative gag polyprotein (gag) and putative envelope glycoprotein (env) genes, partial cds" (accession: GQ497345.1) - it is 99% identical to the 22Rv1 XMRV sequence, with a 100% of query coverage.

5) "Xenotropic MuLV-related virus clone WPI-1106 putative gag-pro-pol polyprotein (gag-pro-pol), putative gag polyprotein (gag), and putative envelope glycoprotein (env) genes, complete cds" (accession: GQ497344.1) - at first it says that they are 100% identical with 100% of query coverage, but when you scroll down you see that they are only 99.902% identical (8160 identical nucleotides out of 8168). For example, in nucleotide number 7401 of the WPI sequence there is a "G", while in the compared nucleotide (number 7441) there is an "A".

6) "Xenotropic MuLV-related virus clone WPI-1178 putative gag-pro-pol polyprotein (gag-pro-pol), putative gag polyprotein (gag), and putative envelope glycoprotein (env) genes, complete cds" (accession: GQ497343.1) - at first it says that it's 100% identical with a query coverage of 100%, but when you scroll down you see that it's just 99.963% identical (8165 identical nucleotides out of 8168).
« Last Edit: March 09, 2011, 07:02:33 PM by omerbasket »

subtr4ct

  • Hero Member
  • *****
  • Posts: 945
  • Just came to freak you out, baby
From the Paprotka CROI abstract, it seems (there is some imprecision due to rounding error) that preXMRV1 and preXMRV2 differ from VP62 by 7 nucleotides, while their hypothetical replication competent recombinant would differ by 5 nucleotides.

Does recombination have to be invoked to explain this?  Couldn't mutation get preXMRV 2 nucleotides closer to VP62 (or 7 nucleotides closer)?  Is it possible or even likely that these murine ERVs would spontaneously go exogenous, even in the absence of xenografting?  Perhaps much earlier than 30 years ago?
Disclaimer: I am not a medical doctor.  This post is not medical advice.  Consult your physician before taking any action.

Gerwyn

  • Guest
"Dr. Yes", as you requested, I've checked for the results of comparing the published WPI sequences to the proviral XMRV from 22Rv1 full genome. These are the results:

1) "Xenotropic MuLV-related virus isolate WPI-1169 putative polyprotein gene, partial cds" (accession: GQ483510.1) - it is 98% identical to the proviral XMRV from 22Rv1 full genome, with a 99% of query coverage.

2) "Xenotropic MuLV-related virus isolate WPI-1138 putative polyprotein gene, partial cds" (accession: GQ483509.1) - it is 99% identical to the 22Rv1 XMRV sequence, with a 99% of query coverage.

3) "Xenotropic MuLV-related virus isolate WPI-1130 putative polyprotein gene, partial cds" (accession: GQ483508.1) - it is 99% identical to the 22Rv1 XMRV sequence, with a 99% of query coverage.

4) "Xenotropic MuLV-related virus clone WPI-1104 putative gag polyprotein (gag) and putative envelope glycoprotein (env) genes, partial cds" (accession: GQ497345.1) - it is 99% identical to the 22Rv1 XMRV sequence, with a 100% of query coverage.

5) "Xenotropic MuLV-related virus clone WPI-1106 putative gag-pro-pol polyprotein (gag-pro-pol), putative gag polyprotein (gag), and putative envelope glycoprotein (env) genes, complete cds" (accession: GQ497344.1) - at first it says that they are 100% identical with 100% of query coverage, but when you scroll down you see that they are only 99.902% identical (8160 identical nucleotides out of 8168). For example, in nucleotide number 7401 of the WPI sequence there is a "G", while in the compared nucleotide (number 7441) there is an "A".

6) "Xenotropic MuLV-related virus clone WPI-1178 putative gag-pro-pol polyprotein (gag-pro-pol), putative gag polyprotein (gag), and putative envelope glycoprotein (env) genes, complete cds" (accession: GQ497343.1) - at first it says that it's 100% identical with a query coverage of 100%, but when you scroll down you see that it's just 99.963% identical (8165 identical nucleotides out of 8168).

remember we are only talking about gag sequences for 22Rv1 because the whole genome has not been fully sequenced

can anyone do a blast search for the env regions

Dr. Yes

  • Hero Member
  • *****
  • Posts: 1103
Hi omer et al,

I was able to do a tiny bit of BLASTing but only a little analysis as I am totally blasted myself!  Actually all I can do is raise a few questions about BLAST sequence comparisons as presented by this website tool.  As my brain is a large fried egg at the moment, I'm not sure if any of the following will be clear, or even of any use!

There is something I can't figure out at nt 152 on the Lo/Alter fragment omer posted about (accession # HQ601957.1); most of the subject sequences have a lengthy break in the alignment there (you can see it on the red graphical sequence schematics at the top of the page; it appears as a black line in the middle of the sequence).  Anyone know what that means?  Are they omitting any non-identities that don't align, thus inflating the percentage identity estimate?  Not sure.

Leaving aside that point, there is indeed a lot of homology between this Lo/Alter gag fragment and 22Rv1, VP62, etc; nearly 100%, as omer pointed out.  Keep in mind, though, that this Lo/Alter fragment is small (only 276 bp) and comes from a region of gag further downstream than the beginnings of the larger Lo/Alter gag fragments.

Also, compare the BLAST results for the larger (698 bp) Lo/Alter fragment "MLV-related virus CFS isolate CFS-type1 polyprotein gene, partial cds" (accession # HM630562.1): notice that it has less overall percent identity (96%) with XMRV isolates like 22rv1 and VP62, and seems to start after the 24 bp gag leader sequence deletion.*  If you look at the same gag region from which the fragment HQ601957.1 was taken (and had close to 100% identity in sequence alignment with prostate XMRV isolates), you do find that HM630562.1 has only 95-96% percent identity with aligning sequences of VP62 and 22Rv1, respectively, which supports the idea that the smaller fragment could come from a virus that maintains high percent identity over a longer gag range...

What all this means is that there is at least one Lo/Alter gag frag that is highly similar to prostate isolates, though it covers only a small region of gag.  However, at least one of their larger isolated fragments is less similar.

It would be interesting to compare the other Lo/Alter sequences to the prostate and WPI sequences as well.  As G just pointed out, the env sequences would be interesting, in part because of the higher variability one might expect there under selective pressure from the immune system.

*Note: I think that the BLAST percentage identity estimates are leaving out the 24 bp deletion of "classic" XMRV isolates, since these don't align with the Lo/Alter fragments; it seems that percent identity is based on similarities in parts of the sequence that align, and does not incude those that do not.  If that deletion were included, the genetic homology would of course be lower (by about 2-3% in terms of percent identity over the 698 bp gag region).  I am not sure if and when deletions are considered in sequence homology calculations, however.
-"Remember what the doctor said?"
 
-"Of course: Grandpa Seth is an invention of my subconscious."

JT1024

  • Hero Member
  • *****
  • Posts: 1861
I know I'm not up to date nor do I have the capability to analyze or evaluate the "fine details" of all the studies or the "BLAST" performed by omerbasket or analyzed by the good doc above, but I'm thinking that Lo/Alter/Ruscetti are not sitting by idly while Coffin/Stoye and company chant "contaminant".

As I've mentioned previously, the vernacular of the retrovirologist/molecular biologist utilize and interpret terms differently than the general public do.

"The contaminants", aka XMRV/PMLV, still appear to be reproductive competent retroviruses and while Coffin/Stoye etc are using this term in their context, I want them to answer questions regarding the pathology and epidemiology of this retrovirus that has resulted in an immune response among ME/CFS patients. An immune response occurs when our immune systems encounter something foreign that needs to be neutralized. 

What is their response to the presence of antibodies to XMRV/PMLV's?  If none of the cell lines/reagents/testing procedures/labs utilized culture or assay material that could potentially "produce" XMRV/PMLV's, then what to they attribute the significant difference between controls and ME/CFS patients?

I hope someone can update me.... I've been offline for most of the last 12 hours.

~ JT
First they Ignore you , then they Laugh at you , then they Attack you , then you WIN!!!

Mahatma Gandhi

Gerwyn

  • Guest
Hi omer et al,

I was able to do a tiny bit of BLASTing but only a little analysis as I am totally blasted myself!  Actually all I can do is raise a few questions about BLAST sequence comparisons as presented by this website tool.  As my brain is a large fried egg at the moment, I'm not sure if any of the following will be clear, or even of any use!

There is something I can't figure out at nt 152 on the Lo/Alter fragment omer posted about (accession # HQ601957.1); most of the subject sequences have a lengthy break in the alignment there (you can see it on the red graphical sequence schematics at the top of the page; it appears as a black line in the middle of the sequence).  Anyone know what that means?  Are they omitting any non-identities that don't align, thus inflating the percentage identity estimate?  Not sure.

Leaving aside that point, there is indeed a lot of homology between this Lo/Alter gag fragment and 22Rv1, VP62, etc; nearly 100%, as omer pointed out.  Keep in mind, though, that this Lo/Alter fragment is small (only 276 bp) and comes from a region of gag further downstream than the beginnings of the larger Lo/Alter gag fragments.

Also, compare the BLAST results for the larger (698 bp) Lo/Alter fragment "MLV-related virus CFS isolate CFS-type1 polyprotein gene, partial cds" (accession # HM630562.1): notice that it has less overall percent identity (96%) with XMRV isolates like 22rv1 and VP62, and seems to start after the 24 bp gag leader sequence deletion.*  If you look at the same gag region from which the fragment HQ601957.1 was taken (and had close to 100% identity in sequence alignment with prostate XMRV isolates), you do find that HM630562.1 has only 95-96% percent identity with aligning sequences of VP62 and 22Rv1, respectively, which supports the idea that the smaller fragment could come from a virus that maintains high percent identity over a longer gag range...

What all this means is that there is at least one Lo/Alter gag frag that is highly similar to prostate isolates, though it covers only a small region of gag.  However, at least one of their larger isolated fragments is less similar.

It would be interesting to compare the other Lo/Alter sequences to the prostate and WPI sequences as well.  As G just pointed out, the env sequences would be interesting, in part because of the higher variability one might expect there under selective pressure from the immune system.

*Note: I think that the BLAST percentage identity estimates are leaving out the 24 bp deletion of "classic" XMRV isolates, since these don't align with the Lo/Alter fragments; it seems that percent identity is based on similarities in parts of the sequence that align, and does not incude those that do not.  If that deletion were included, the genetic homology would of course be lower (by about 2-3% in terms of percent identity over the 698 bp gag region).  I am not sure if and when deletions are considered in sequence homology calculations, however.

yes they are looking at the sequence homology of the "bits" that have been sequenced  very little of the 22RV1 genome has been sequenced henceth e fact that is the same as wild type XMRV is a hypothesis

Gerwyn

  • Guest
I know I'm not up to date nor do I have the capability to analyze or evaluate the "fine details" of all the studies or the "BLAST" performed by omerbasket or analyzed by the good doc above, but I'm thinking that Lo/Alter/Ruscetti are not sitting by idly while Coffin/Stoye and company chant "contaminant".

As I've mentioned previously, the vernacular of the retrovirologist/molecular biologist utilize and interpret terms differently than the general public do.

"The contaminants", aka XMRV/PMLV, still appear to be reproductive competent retroviruses and while Coffin/Stoye etc are using this term in their context, I want them to answer questions regarding the pathology and epidemiology of this retrovirus that has resulted in an immune response among ME/CFS patients. An immune response occurs when our immune systems encounter something foreign that needs to be neutralized. 

What is their response to the presence of antibodies to XMRV/PMLV's?  If none of the cell lines/reagents/testing procedures/labs utilized culture or assay material that could potentially "produce" XMRV/PMLV's, then what to they attribute the significant difference between controls and ME/CFS patients?

I hope someone can update me.... I've been offline for most of the last 12 hours.

~ JT

Jt-you have asked questions about the science involved---they would probably redefine that as "cheating"

omerbasket

  • Hero Member
  • *****
  • Posts: 575
Hi omer et al,

I was able to do a tiny bit of BLASTing but only a little analysis as I am totally blasted myself!  Actually all I can do is raise a few questions about BLAST sequence comparisons as presented by this website tool.  As my brain is a large fried egg at the moment, I'm not sure if any of the following will be clear, or even of any use!

There is something I can't figure out at nt 152 on the Lo/Alter fragment omer posted about (accession # HQ601957.1); most of the subject sequences have a lengthy break in the alignment there (you can see it on the red graphical sequence schematics at the top of the page; it appears as a black line in the middle of the sequence).  Anyone know what that means?  Are they omitting any non-identities that don't align, thus inflating the percentage identity estimate?  Not sure.

Leaving aside that point, there is indeed a lot of homology between this Lo/Alter gag fragment and 22Rv1, VP62, etc; nearly 100%, as omer pointed out.  Keep in mind, though, that this Lo/Alter fragment is small (only 276 bp) and comes from a region of gag further downstream than the beginnings of the larger Lo/Alter gag fragments.

Also, compare the BLAST results for the larger (698 bp) Lo/Alter fragment "MLV-related virus CFS isolate CFS-type1 polyprotein gene, partial cds" (accession # HM630562.1): notice that it has less overall percent identity (96%) with XMRV isolates like 22rv1 and VP62, and seems to start after the 24 bp gag leader sequence deletion.*  If you look at the same gag region from which the fragment HQ601957.1 was taken (and had close to 100% identity in sequence alignment with prostate XMRV isolates), you do find that HM630562.1 has only 95-96% percent identity with aligning sequences of VP62 and 22Rv1, respectively, which supports the idea that the smaller fragment could come from a virus that maintains high percent identity over a longer gag range...

What all this means is that there is at least one Lo/Alter gag frag that is highly similar to prostate isolates, though it covers only a small region of gag.  However, at least one of their larger isolated fragments is less similar.

It would be interesting to compare the other Lo/Alter sequences to the prostate and WPI sequences as well.  As G just pointed out, the env sequences would be interesting, in part because of the higher variability one might expect there under selective pressure from the immune system.

*Note: I think that the BLAST percentage identity estimates are leaving out the 24 bp deletion of "classic" XMRV isolates, since these don't align with the Lo/Alter fragments; it seems that percent identity is based on similarities in parts of the sequence that align, and does not incude those that do not.  If that deletion were included, the genetic homology would of course be lower (by about 2-3% in terms of percent identity over the 698 bp gag region).  I am not sure if and when deletions are considered in sequence homology calculations, however.
I have just looked at that again and you're right about the deletion - for some reason, the deletion is not even written in the sequences in NCBI. The sequences just continues with the letter that is after the deletion, without showing that there is a deletion.

However, I've done some manual work: at the supporting material for the Lo/Alter study (http://www.pnas.org/content/suppl/2010/08/16/1006901107.DCSupplemental/pnas.201006901SI.pdf#nameddest=STXT starting at page 3), there is a comparison between sequences found by them to other sequences. So I've manualy compared their "CFS type-1_18 members" sequence to VP62 as they show it there. Including a 15 bp deletion (from the 35th nucleotide to the 49th nucleotide), which is the only deletion in XMRV that does not appear in "CFS type-1_18 members", the "CFS type-1_18 members" sequence has 710 identical nucleotides (in the right place, ofcourse) to the VP62 sequence. Since the "CFS type-1_18 members" sequence has 748 nucleotides, it means that it is 710/748*100=94.92% identical to VP62. As I said earlier - there are HIV-1 full sequences that are only 89% identical to other HIV-1 full sequences. If I'm not mistaken, in another rather quick, search, I've also seen HIV-1 full sequences that are only 85% identical to other HIV-1 full sequences.

So why when it comes to HIV-1 2 viruses that are "just" 85% identical can be called by the same name, but when it comes to XMRV two viruses that are 94.92% identical cannot be called by the same name?

It's exactly what Dr. Alter said to Mindy Kitei: HCV is also not one virus. It's a whole bunch of viruses that are similar enough to be called by the same name.
« Last Edit: March 10, 2011, 08:46:19 PM by omerbasket »

omerbasket

  • Hero Member
  • *****
  • Posts: 575
Looking at the envelope sequences gave "only" 84% similarity between HQ157343.1 and VP62, VP42, vP35, the WPI's full sequences and the 22Rv1 sequence (with a query coverage of 100%), 86% similarity between HQ157342.1 and the sequences described above (VP62 etc.) with a query coverage of 100%, and HQ157342.1 showed 85% similarity to the described sequences (with a query coverage of 100%).  We should remember that the envelope sequences there are pretty small - one (the 86% one) is 206 nucleotides, the two others are 240 nucleotides each.

Altough it's a little less similar than the 748 nucleotides sequence, it seems to me that it's identical enough (since for HIV-1 they say 85% is identical enough), and correct me if I'm wrong, but I think that the longer sequence (the 748 nuclotides one that I described in my last post) would represent better the similarity between the sequences found in the Lo/Alter study and XMRV, right?

belcanto

  • Hero Member
  • *****
  • Posts: 1029
good memory, fly - i had forgotten that.  Sure looked like a deliberate interruption!

When Judy M. talks about the xmrv that she has "sequenced" and says that she wants to sequence a lot more, is that different than sequencing the "entire genome"?


JT1024

  • Hero Member
  • *****
  • Posts: 1861
Omerbasket, Dr. Yes, ?? Gerwyn, and whoever else is into "blasting" and comparisons between XMRV/PMLV's and HIV, the "contaminant theorists" have been playing words games with us. 

The Weasels, Reeves, McClures, Coffin, Stoye, etc are no match for Alter, Ruscetti, Lo, Mikovits, Montaigner, Bell, etc.  The way Coffin and Stoye speak, you would think they were the only retrovirologists in the world. Did either discover HIV? HTLV? Hep B? HEp C? 

IMHO...GAME OVER... or almost over.

I admire all your abilities and extensive knowledge!  I look forward to learning much more but will never reach your level. I'm too much a generalist.. pretty good at a lot, great at nothing.. but able to see the big picture, interrelationships, and opportunities. 

From what I've "heard" online, it seems public acknowledgement that the "new" retrovirus (XMRV) is imminent. Imagine the planning required behind closed doors trying to not freak everyone out.

Love to hear the geek speak and glimpse over your shoulders while you examine the evidence. Hopefully you won't mind me expressing myself periodically when I have a thought that may or may not be relevant! 

Let's hope for great news soon!  ~ JT
First they Ignore you , then they Laugh at you , then they Attack you , then you WIN!!!

Mahatma Gandhi

omerbasket

  • Hero Member
  • *****
  • Posts: 575
I cant remem totally,

any one no where it is, i think their were transcripts done. I can't even remem when it was.

it was our first introcution to stoye, was it first international XMRV worhsop?
Yes, you can find it on youtube, starting here:
1st International Workshop on XMRV Q A Wrap-up Session2.flv

And JT, please feel free to "interrupt" whenever you want to, since you're not interrupting at all!

JT1024

  • Hero Member
  • *****
  • Posts: 1861
That was such a painful panel to watch.  Stoye needs to go back to London.

What on earth was the NIH thinking in selecting Stoye as a moderator of that session?
First they Ignore you , then they Laugh at you , then they Attack you , then you WIN!!!

Mahatma Gandhi

omerbasket

  • Hero Member
  • *****
  • Posts: 575
That was such a painful panel to watch.  Stoye needs to go back to London.

What on earth was the NIH thinking in selecting Stoye as a moderator of that session?
I think that Coffin was very strong at organizing this workshop - and he seems to adore Stoye.