Long time readers of ERV know that I do not believe a ‘new’ retrovirus, XMRV, is the causative agent of any human disease. It does not make sense as a real human pathogen, unless you disregard field basics (or make up new ‘rules’).
New cell transformation/cancer rules.
New transmission/epidemiology rules.
New immunology rules.
New virology rules.
New genetics/population biology/evolution rules.
Over and over and over with XMRV, Ive typed in frustration “THIS DOES NOT MAKE SENSE!!!!!”
Occams Razor, though not always useful in science, does provide us with a basic logical guide: Either XMRV is a real human pathogen that requires the upheaval of >decades of basic biology… or XMRV is a contaminant.
Its probably the latter, but that is just pure logic. Not science. Reality and logic do not always go hand-in-hand, especially with biology. Biology is a messy, silly process sometimes (see: evolution), and XMRV might indeed be a messy, silly virus that behaves bizarrely in humans. I can scream “THIS DOES NOT MAKE SENSE!!!!!” all I want, and it might not, but it still might be true.
While I was gone, a series of papers were published in Retrovirology that gave experimental support for the logical conclusion that XMRV is a contaminant:
Contamination of clinical specimens with MLV-encoding nucleic acids: implications for XMRV and other candidate human retroviruses (review)
Disease-associated XMRV sequences are consistent with laboratory contamination (blagged here)
An endogenous murine leukemia viral genome contaminant in a commercial RT-PCR Kit is amplified using standard primers for XMRV
Contamination of human DNA samples with mouse DNA can lead to false detection of XMRV-like sequences
Mouse DNA contamination in human tissue tested for XMRV
What does this research mean?
Its a collection of putative explanations for confusing results:
Why you can ‘find’ XMRV in some labs, and not others, in different parts of the world. Different people use different reagents. Its as variable as what kind of pop people like to drink. My labmate likes Superscript III for reverse transcription. I prefer a different brand for the same damn process. The lab next door might use something different. A lab in Germany might use something different. One of the papers showed how a reagent used in the ‘XMRV positive’ papers is absolutely contaminated by mouse DNA, thus can absolutely give false positive results via mouse ERVs. Other reagents popular in other labs were not contaminated (‘XMRV negative’). Does this mean that all of the ‘XMRV positive’ papers that used the contaminated reagent were also contaminated and should be retracted? No. It means that this is a potential explanation for the discrepancies in XMRV positive/negative labs, and the pos labs must investigate this before they publish again.
Contamination can happen at collection. PCR reagents are not the only way you can get false positives. Another paper showed that their own samples, the actual samples, had mouse DNA in them. The second they were collected, they were contaminated, and they showed exactly what kind of test you need to perform to make sure that future samples are not likewise contaminated. Does this mean that pos papers were also contaminated prior to any other manipulation? No. It means that post labs must perform this test in the future to demonstrate that they dont have contaminated samples at the starting gate.
Cell lines are contaminated. I do HIV-1 research. When I want to do experiments in the lab, I can use primary cells, or cell lines. Primary cells would be like CD4+ T-cells purified from blood donated at a blood bank. Yes, they are very biologically relevant (they ARE the cells HIV-1 infects), but there are some problems. Primary cells are not all the same (from the same person, or from different people), they only live a couple of weeks, you can only get so many from a patient, meh. Another option is using cell lines. ‘Immortal’ (continually dividing, not literally immortal) pretty much identical cells. Not exactly biologically relevant (like my U87 cell line is actually glial cells forced to express CD4 and CCR5 or CXCR4– HIV aint normally infecting glial cells), but good for consistency and quantity. Another problem? Cell lines can have a stage in their development in mice. This can lead to contamination with mouse viruses, like what happened to TZM-bls. Turns out MLV-like-viruses are in lots and lots of cell lines, and could be a huge potential source of false positives. This might also explain why the WPIs archaic-yet-proclaimed-better culture ‘test’ (I really cant emphasize enough how outdated viral culture is in 2010) is ‘better’– the longer you culture cells, the more you increase the contaminant false positive signal.
PCR protocol can effect detection of contamination. Okay, lets say you have a sample. It might be contaminated with mouse DNA. You work in a lab that might be contaminated with mouse viruses. Youve got PCR reagents you bought from the store. It might be contaminated with mouse DNA. Is there any way you can detect XMRV, and ignore all the contamination? Maybe. You use primers that are specific for what youre looking for, and will ignore stuff that kinda looks like what you are looking for, if you can. You must (at least) look for mouse IAPs as a marker for contamination (not mitochondrial DNA). You do not do what the ‘XMRV positive’ papers did and use nested PCR, which will amplify even the tiniest bits of anything that kinda looks like what you are looking for. Does this mean that the ‘XMRV positive’ papers amplified non-specific crap? No. It means they need to be extraordinarily careful in their primer design, and should not just use a primer set/PCR method that someone else used just because someone else used it. You must also extensively study the amplified sequence to ensure that the sequence is real (see below).
The stuff being sequenced is almost certainly crap. You cannot just sequence your ‘new’ virus and declare ‘Mission Accomplished’. You cannot make value claims like ‘there was sequence evolution’ or ‘there is sequence diversity’ based off of eye-ball ‘measurements’ of your sequence. You need to collect your data and properly analyze it– extensively. Or someone else will do it. And your ‘eye-ball’ might not match reality. This particular criticism I do lay at the feet of the ‘XMRV positive’ paper authors who made claims without the science to back them up. Particularly frustrating were these sequences very obvious homology to endogenous retroviruses, yet absolutely no serious investigation into contamination. XMRV bizarrely did not behave as a quasispecies, unlike every other retrovirus on the planet, yet no one appeared to be interested in investigating this astounding ‘fact’. Shocking.
What are the long-term effects of this research? Does it ‘prove’ XMRV isnt real?
Its a bullshit filter.
Experimental evidence for Occams Razor– XMRV is a contaminant, not a real human pathogen.
Because this is now published information, authors of ‘XMRV pos’ papers can no longer harumph ‘ITS NOT CONTAMINATION!’ to the press while doing no investigation into contamination. It is now part of the literature that reagents, mouse DNA, and viral RNA/DNA can confound results, and that sequence analysis supports this. You cannot publish an XMRV positive paper without now investigating and directly addressing all of this anymore (unless you publish in PNAS, I suppose…).
Contamination cannot be side-stepped or ignored anymore.