New WPI and CDC XMRV sequences in genbank

[ukxmrv]

At the conference it can be hard to get the microphone to ask q's. What I normally so is write them down clearly before (if I can) and then pass them to Richard or the person with the mic. He can then pass to the right doctor at the right Q&A part.

Hope that helps.

 

[omerbasket]

OK so we have a variable retrovirus in prostate cancer, and one that isnt varying enough in ME/CFS!

I'm not sure it's correct to say that. The sequences that vary more from VP62 that Switzer found are not gag sequences, and the sequences published by the WPI this May are only gag sequences. Now, when you look at the only gag sequence published by Switzer you can see that it is 367/373 base paits identical to VP-62, meaning that it is 98.39% identical to VP-62.
One of the WPI's gag sequences is 98.52% identical to VP-62, similar to the percentage found by Switzer. Had he published more than one gag sequence, perhaps we could have been wiser.

The other sequences of the WPI are more identical to VP-62 - but one should notice the following fact:
In the "Science" paper, the WPI sequenced two full-length XMRV and one gag sequnece. The gag sequence was 100% identical to the relative area in VP-62, with a query coverage of 99%. The two full length sequences were 99.73% and 99.94% identical to VP-62. Now, the WPI has published sequences that some of them are very similar (mostly about 99.7% identical to VP-62), but some of them are 99.14%, 99.18% and 98.52% (as I've mentioned) identical to the relative area in VP-62. So, that is some more variation from what they found at first.

Now, if that little variation should tell us that it is a contamination, then why did no one, including Coffin (which seemed to be enthusiastic about the findings at the beginning) mention this after the original "Science" paper was published, and before the negative papers were published? Perhaps because they thought that it is possible?

So, what's changed? It is still as possible as it was before. And not only that - but now we also have sequences that despite still being very similar to VP-62, are significantly less similar to it than the ones found in the original "Science" paper.

"currer", one thing I'd be glad if you'd ask Dr. Mikovits is whether they are about to publish more sequences soon. I tend to think that they are about to do that, and also, it's very possible that they have already submitted more sequences but those hasn't been approved for publication yet (by NCBI).

 

[currer]

OK. omer, I will submit some questions about this for PR.

I dont know anymore what to make of all this. Frank Ruscetti is no fool, didn't he work with Gallo on HIV? The WPI ought to know contamination if they have it. I do wish things were clearer. Perhaps after tomorrow I will know one way or the other.
JM will be there and I may be able to assess the whole situation more accurately in real life.

By the way one of my previous posts here was wrong about the Switzer paper's findings. I've edited it but I cant change the text where my post has been quoted.
This will teach me to check carefully before posting in future!

 

[omerbasket]

I think the following quote from a thing that Gerwyn wrote on another forum might be important:

The env region is the most hypervariable some of theGAG isolates have a 2% max identity variation from VP62 in a 300 base sequence from an 8 kilobase genome this may not be apparent at first but a PCR based on vp62 using high stringency primers would easily miss these sequences.

It fits with what I've said - that the variability that Switzer got was mostly from the envelope sequences, while the WPI's newly-published sequences are only gag sequences - and I've shown you that they have one gag sequence that is very close to the variability gotten by Switzer.

And the second line of Gerwyn is also very important - and again, we go back to annealing temperatures, magnesium and other stuff - and that is why we need a full replication.

 

[kday]

In the "Science" paper, the WPI sequenced two full-length XMRV and one gag sequnece. The gag sequence was 100% identical to the relative area in VP-62, with a query coverage of 99%. The two full length sequences were 99.73% and 99.4% identical to VP-62. Now, the WPI has published sequences that some of them are very similar (mostly about 99.7% identical to VP-62), but some of them are 99.14%, 99.18% and 98.52% (as I've mentioned) identical to the relative area in VP-62. So, that is some more variation from what they found at first.

I went back to the original Science paper, and you are absolutely right! I'm sorry if I posted misinformation and confused everyone.

You can ignore the posts by myself and LJS.

 

[omerbasket]

Thanks, kday!
I also realised that I made a small mistake (but the correction would just strengthen my argument) - one of the full sequences the WPI found was 99.94% identical to VP-62, and not 99.4% as I've written earlier.
Besides that, they also submitted, a very short time after the publication of the "Science" study, two more sequences - these are sequences of "putative polyprotein", and those sequences (which are rather small - both of them have 366 base pairs) are 100% identical to the relevant part in VP-62.

 

[acer2000]

And the second line of Gerwyn is also very important - and again, we go back to annealing temperatures, magnesium and other stuff - and that is why we need a full replication.

This. Can someone ask her to spell out explicitly once and for all what the differing lab conditions have been between all these papers? People have gone over and over the regions targeted by the various PCR tests, but for all we know these other things (temp, mag, etc..) are just as if not more important.

If someone put out a spreadsheet/table going through each study and accounting for all of these other variables, it would go a long way towards reconciling them.

The lack of cooperation on this stuff is maddening.

 

[Otis]

I'm not sure it's correct to say that. The sequences that vary more from VP62 that Switzer found are not gag sequences, and the sequences published by the WPI this May are only gag sequences. Now, when you look at the only gag sequence published by Switzer you can see that it is 367/373 base paits identical to VP-62, meaning that it is 98.39% identical to VP-62.
One of the WPI's gag sequences is 98.52% identical to VP-62, similar to the percentage found by Switzer. Had he published more than one gag sequence, perhaps we could have been wiser.

The other sequences of the WPI are more identical to VP-62 - but one should notice the following fact:
In the "Science" paper, the WPI sequenced two full-length XMRV and one gag sequnece. The gag sequence was 100% identical to the relative area in VP-62, with a query coverage of 99%. The two full length sequences were 99.73% and 99.94% identical to VP-62. Now, the WPI has published sequences that some of them are very similar (mostly about 99.7% identical to VP-62), but some of them are 99.14%, 99.18% and 98.52% (as I've mentioned) identical to the relative area in VP-62. So, that is some more variation from what they found at first.

Now, if that little variation should tell us that it is a contamination, then why did no one, including Coffin (which seemed to be enthusiastic about the findings at the beginning) mention this after the original "Science" paper was published, and before the negative papers were published? Perhaps because they thought that it is possible?

So, what's changed? It is still as possible as it was before. And not only that - but now we also have sequences that despite still being very similar to VP-62, are significantly less similar to it than the ones found in the original "Science" paper.

"currer", one thing I'd be glad if you'd ask Dr. Mikovits is whether they are about to publish more sequences soon. I tend to think that they are about to do that, and also, it's very possible that they have already submitted more sequences but those hasn't been approved for publication yet (by NCBI).

Glad to see more folks running BLAST and related tools....

I think it would be worth asking if additional full-length sequences might be on the way.

Sent from my ADR6400L using Tapatalk

 

[omerbasket]

This. Can someone ask her to spell out explicitly once and for all what the differing lab conditions have been between all these papers? People have gone over and over the regions targeted by the various PCR tests, but for all we know these other things (temp, mag, etc..) are just as if not more important.

If someone put out a spreadsheet/table going through each study and accounting for all of these other variables, it would go a long way towards reconciling them.

The lack of cooperation on this stuff is maddening.

The differences are very technical - I don't know if she would remember the exact differeneces just from her head. One difference, for example, is using a kit of one company instead of a similar kit of another company. It seems unimportant - but it might be important.

 

[acer2000]

The differences are very technical - I don't know if she would remember the exact differeneces just from her head. One difference, for example, is using a kit of one company instead of a similar kit of another company. It seems unimportant - but it might be important.

Sure... but at this stage, given the inconsistencies that have plagued testing, doesn't it behoove them to sort out these subtle differences in testing conditions and methodology?

 

[currer]

Hi, I got back late last night from the Invest in ME conference.

Some of the studies that were presented have not been published yet, so we were asked to maintain confidentiality by not posting about them. I initially understood this embargo to cover only one piece of research presented, but I think it certainly covers more than that, so I wont say any more at the moment.
Neither of these two areas were specifically to do with work coming out of the WPI.

The presentation from the WPI by Judy Mikovits was a similar talk to her recent ones, focusing on how they are looking for new polytropic variants of XMRV in the blood.
I did put a question forward about our concerns on this thread, but due to time constraints it did not get asked. However, Dr. Mikovits did say they have further sequences to publish in Genbank.

There are positive and exciting new areas of research happening, so people can be very hopeful for the future. The WPI are confident in their findings and Judy Mikovits looks as if she is thriving on all the work.

 

[ukxmrv]

I put forward a question about the new sequences in gene-bank but it didn't fully get asked. My handwriting is so bad that I had to do it 4 times until it looked legible and that was before the conference (my printer died on me a few days ago).
Dr Mikovits said that the sequences confirmed the variety that was more like Dr Lo's. There were more polytropic variants.
That's changes in individuals didn't have to be consistent. She suspected that in some patients the isolate would be very close 40 years later (she said this is common in HTLV).
Dr Mikovits said that HIV is only a model for HIV.

p.s. earlier in the session Dr Mikovits said that Dr Coffin would be publishing a paper shortly that would shed light on the origins of XMRV (or something like that I'm pretty wobbly today)

.

 

[Bob]

Thanks for the feedback, currer & ukxmrv.
Hope you enjoyed yourselves at the conference.
I'm looking forward to watching the DVD.

 

[insearchof]

I think the following quote from a thing that Gerwyn wrote on another forum might be important:


It fits with what I've said - that the variability that Switzer got was mostly from the envelope sequences, while the WPI's newly-published sequences are only gag sequences - and I've shown you that they have one gag sequence that is very close to the variability gotten by Switzer.

And the second line of Gerwyn is also very important - and again, we go back to annealing temperatures, magnesium and other stuff - and that is why we need a full replication.

Hi Omerbasket

An obscure question for you or anyone else who might know.....

What role does magnesium play in the process and when and how is it added?

 

[alex3619]

An obscure question for you or anyone else who might know..... What role does magnesium play in the process and when and how is it added?

Hi insearchof,

From wikipedia: http://en.wikipedia.org/wiki/Polymerase_chain_reaction_optimization#Magnesium_concentration

Magnesium concentration

Magnesium is required as a co-factor for thermostable DNA polymerase. Taq polymerase is a magnesium-dependent enzyme and determining the optimum concentration to use is critical to the success of the PCR reaction.[3] Some of the components of the reaction mixture such as template concentration, dNTPs and the presence of chelating agents (EDTA) or proteins can reduce the amount of free magnesium present thus reducing the activity of the enzyme.[4] Primers which bind to incorrect template sites are stabilized in the presence of excessive magnesium concentrations and so results in decreased specificity of the reaction. Excessive magnesium concentrations also stabilize double stranded DNA and prevent complete denaturation of the DNA during PCR reducing the product yield.[3][4] Inadequate thawing of MgCl2 may result in the formation of concentration gradients within the magnesium chloride solution supplied with the DNA polymerase and also contribute to many failed experiments.[4]


Back to me again - what does this mean? The polymerase used is stable at very high temperatures (it is derived from an organism that lives in extremely hot water), and those temperatures alter the physical characteristics of the DNA, speparating the twin strands, which makes PCR possible. The polymerase enzyme is magnesium dependent, it can't copy DNA without magnesium. Too little magnesium means the polymerase will not work, too much protects the DNA even so that it is stable at high temperatures - it remains double stranded. Double stranded DNA cannot be copied without first separating the strands.

I gather that optimization of magnesium concentrations is dependent on the target DNA. I do not know enough about this process to comment further on this point, I would only be speculating.


http://www.caister.com/molecular-biology-blog/2008/11/pcr-troubleshooting-mg-concentration.html

This site discusses Mg troubleshooting for PCR briefly. When certain other substances are used in the reaction, they can alter magnesium ion concentrations and so the magnesium level has to be adjusted. There are entire manuals on PCR optimization online, and a number of books.

I have never used a PCR machine, although I was involved in labwork in which PCR was used, but the machine was handled by an experience operator. My knowledge is more theoretical, and now old - there are many new types of PCR reactions than I studied when I was at university.

Of interest in the XMRV debate is that reverse transcription (RT-PCR) is often using MuLV reverse transcriptase, which is one reason that contamination testing is required for all reactions I suppose. RT-PCR is for amplifying RNA rather than DNA - and XMRV is an RNA virus. So if you are looking for the virus, and not its integrated DNA form, you need to look for RNA.

As to when and how it is added, I can only speculate. It is probably added at the very beginning with the rest of the reaction mixture. I doubt any is added later, if for no other reason than this would increase risk of contamination. However, this is an engineering issue, and different machines may or may not allow this - I do not know.

Bye
Alex

 

[ukxmrv]

There was something said at the conference yesterday about magnesium. Along the lines that the right tubes are needed for blood collection in the XMRV studies. I can't remember if this was said in relation to the failed studies or the BWG. Something about heparin tubes needed as the EDTA tubes leech the magnesium out of the LNcap cells (that is used for the culture). This reduces efficiency. Sorry, brain fried and typing from now cryptic written notes.

 

[currer]

Hi, As I explained yesterday, the IiME conference were very strict about not publicising unpublished research so I cannot say much.

I would just like to say however, that I was very impressed by both Annette Whittemore and Judy Mikovits

I am convinced by Judy Mikovits. I thought she was a highly intelligent and intuitive woman, with an unusual mind. She speaks so quickly because she thinks very rapidly and is drawing together a multitude of ideas as she speaks. She is a very gifted woman, maybe a genius. I would certainly trust her judgement when it comes to virology. In fact I thought she towered over all the other researchers intellectually.

Her high intelligence makes her unusual, and perhaps because they are not her intellectual equivalent, more average people are tempted to mistrust her findings and look for a consensus. Judy Mikovits has the courage and ability to think and judge for herself from her own data. Such people will always be criticised.

Most researchers would not have the intellectual confidence in their work to drive this research forward against such resistance. But she can have it because her grasp of the subject is complete.
I think this is why she is outspoken at times.
She really is in an entirely different league to other people. No wonder she works with Frank Ruscetti.

We should not lose confidence in the WPI. I believe the WPI are right.
But Annette Whittemore did warn us that we have to be prepared for more resistance ahead in getting the work accepted.

 

[toddm1960]

p.s. earlier in the session Dr Mikovits said that Dr Coffin would be publishing a paper shortly that would shed light on the origins of XMRV (or something like that I'm pretty wobbly today)

ukxmrv did Judy have that knowing look her on face meaning coffin now has proff it's not all contamination? Or was it one saying there's more resistance ahead?

 

[currer]

Hi, I assumed this just to be the paper on the prexmrv 1 and 2 and the 22RV1 cell line in the grafted mice. The one presented at the State of knowledge conference. That is unpublished at the moment.

 

[VillageLife]

Yes dr Mikovits said Dr Coffin is a good source for the origin of xmrv, meaning that xmrv comes from the lab recombination, thats what dr coffin says.

So this XMRV probably got out from the lab workers into the public.