Retrovirology - Latest Comments

What were the characteristics of the patient cohort?

Angela Kennedy — 2010-02-22T00:00:00Z

Immediate questions that spring to mind relate to the patient cohort and whether they were similar enough to the patient cohort in the Lombardi et al project.

The Lombardi et al research cohort apparently met criteria for 'ME/CFS' as identified by Carruthers et al, in addition to Fukuda et al. which has been demonstrated to select patients with post-exertional malaise and fatigue, sleep dysfunction, pain, clinical neurocognitive, and clinical autonomic/ neuroimmunoendocrine symptoms (Jason et al).

Furthermore, the WPI give this information about their patient cohort in their supporting online material:

"Their diagnosis of CFS is based upon prolonged disabling fatigue and the presence of cognitive deficits and reproducible immunological abnormalities. These included but were not limited to peturbations of the 2-5A synthetase/RNase L antiviral pathway, low natural killer cell cytotoxicity (as measured by standard diagnostic assyas) and elevated cytokines particularly interleukin-6 and interleukin-8. In addition to these immunological abnormalities, the patients characteristically demonstrated impaired exercise performance with extremely low VO2 max measured on stress testing..." (www.sciencemag.org/cgi/content/full/117905/DC1)

The Erlwein et al project selected a rather different patient cohort:

""Patients in our CFS cohort had undergone medical screening to exclude detectable organic illness".

In any British subsequent research to Lombardi et al, unless the patient cohort selected was similar enough to that in the Lombardi research, it is premature to believe XMRV is not present in the 'CFS' population in Britain.

Ongoing neglect of the importance of establishing a possible ‘CFS’ patient population in Britain, clinically and in research settings, using the Canadian Guidelines, is preventing the development of knowledge that might help extremely ill and disabled people, diagnosed with 'CFS', known to be here in Britain. I am therefore interested if the authors are aware of any serious attempts to identify such a population in Britain, or taking part in such attempts themselves.

The problems I have briefly outlined here do not fully express the range and depths of problems with regard to: the identity of an accurate ‘CFS’ population; the instabilities of ‘CFS’ criteria per se; the faulty concepts of ‘medically unexplained’ or ‘functional‘ and relation to ‘psychogenic‘ explanations for somatic illness; the vagaries of criteria that claim to facilitate a ‘diagnosis of exclusion’; and the psychogenic dismissal of serious organic dysfunction of patients given a ’CFS’ diagnosis, problems that have happened for many years.

REFERENCES

Carruthers, B. et al (2003) “Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Clinical Working Case Definition, Diagnostic and Treatment Protocols” Journal of Chronic Fatigue Syndrome, Vol. 11(1), pp 7 - 115.

Jason LA, Torres-Harding SR, Jurgens A, Helgerson J. “Comparing the Fukuda et al. Criteria and the Canadian Case Definition for chronic Fatigue Syndrome”. Journal of Chronic Fatigue Syndrome 12(1):37-52, 2004

Wrong name

Antonio V Borderia Giner — 2010-01-29T00:00:00Z

Hi,

The name of the first author is wrong. It should be Antonio and not Antonia.

Thanks
Antonio

Addition of authors not included in the original submission to the journal

Independent Journals — 2009-11-24T00:00:00Z

After publication of this work we realised that the full list of authors had not been included. The missing authors were as follows:

Andrei Ogrel
Thanh Le
Catalina Soare
David E. Anderson
Jose Torres
Francisco Diaz-Mitoma

These authors were originally included on the conference abstracts poster and 2009 Aids Vaccine Conference Abstracts book but were not included in the original submission to the journal. All authors have now been added to the published version.

competing interests

Francoise moreau-gachelin — 2009-11-24T00:00:00Z

"The author(s) declare that they have no competing interests"

Tenofovir treatment augments anti-viral immunity against drug-resistant SIV challenge in chronically infected rhesus macaques

Brian n — 2009-11-24T00:00:00Z

Treatment with tenofovir may, therefore, augment antiviral immunity by stimulating immunomodulatory factors and creating an environment less permissive.

================================

brian

pennsylvania drug rehab

Response to “How did these retroviruses spread so widely?”

Thomas Grunwald — 2009-10-07T00:00:00Z

As mentioned in our paper “Unintended spread of a biosafety level 2 recombinant retrovirus” contamination of cell lines with replication competent retroviruses has been repeatedly observed, even in laboratories of experienced retrovirologists. Systematic investigations on the frequency of such contaminations are, to the best of our knowledge, missing. In response to a recommendation of the German Central Commission for Biosafety (ZKBS), that laboratories working with gene-modified organisms should test their cell lines for the presence of squirrel monkey retrovirus (SMRV), 128 of 4279 tested aliquots of cell lines were reported to be positive. Approximately 70 different cell lines from at least 13 different laboratories throughout Germany were positive for SMRV. Since not all aliquots of the same cell line were positive, this indicates that contaminations do occur under commonly employed cell culture techniques.
We agree with Dr. Miller´s assumption, that we have imported one or a few infected cell lines and that the contaminants subsequently spread in our laboratory. However, we reject his insinuation that this has been due to poor cell culture techniques. We are using sterile, disposable tissue culture plastic ware and receive media, fetal calf serum and all other cell culture solutions from common suppliers. Cell cultures are regularly checked for mycoplasma and in the rare case of positivity the cell lines are either disposed off or treated with antibiotics until mycoplasma can no longer be detected. All lab members working with tissue cultures are of course trained not to use the same pipette to split different cell lines or to re-fill a pipette that has been previously used to feed cell lines. At least since 2001, all cell lines are stored in the vapor phase above the liquid nitrogen in plastic cryo tubes with a screw cap. Virus stocks are stored at – 80°C, and not as virus infected cultures in liquid nitrogen tanks.
Thus, it remains unclear, as in most previous reports on retroviral contaminants, how these contaminants spread. Formation of aerosols during the splitting of a contaminated culture and subsequent contamination of cultures splitted in the same tissue culture hood via aerosols should also be considered. Even if this is a rare event, the virus might accumulate over time in several cell lines, since different cell lines are maintained simultaneously in the same laboratory for years, particularly if a contamination is not suspected due to lack of cytopathic effects.
While one can only speculate, how the retroviral contaminants spread so widely, we do know that by regular testing for the contaminants and systematic elimination of all contaminated cultures we were able to maintain our cell cultures free of SMRV and the hybrid retrovirus without other major changes in cell culture techniques and storage.

How did these retroviruses spread so widely?

A Dusty Miller — 2009-09-29T00:00:00Z

Speaking as an investigator who does much work with retroviruses, and as the creator of the pAMS hybrid retrovirus mentioned in this report as having spread promiscuously in multiple cell lines, I find it incredible to see such widespread contamination by retroviruses in the authors' laboratories. Provided the PCR data in Table 3 are correct and do not represent amplification of endogenous retroviral sequences, and because I work with many of the listed cells lines and know them to be free of replication-competent retroviruses, I conclude that most of the spread has occurred in the authors' laboratories. I also know that it is relatively easy to prevent spread of retroviruses in cultured cells by using good cell culture techniques. Presumably one or a few infected cell lines were imported into the lab and provided the source of the subsequent widespread contamination.

First, I would ask whether many of the authors' cell lines are also contaminated with mycoplasma. This would be another sign of poor cell culture technique where organisms secreted by one cell type are transferred to another, probably by transfer of culture medium containing the organisms. This can occur by using the same pipette to feed multiple cell types, or by feeding a contaminated cell type and then using that pipette to get more medium from a bottle that is later used to feed other cells.

Beyond the use of poor cell culture practices, the other worry is transfer of viruses during storage of cells in liquid nitrogen. Liquid nitrogen can enter and leave standard freezing vials, and I have always wondered if it would be possible that frozen virus might be transmitted between cell lines by this route, although I have never detected this. Storage of cells in the vapor phase above the liquid nitrogen should prevent this provided the liquid nitrogen level never rises above the stored vials. Which type of frozen cell storage was used in this case? In my lab we usually freeze virus-producing cells in sealed glass vials to prevent any possibility of virus transmission, especially when dealing with pathogenic retroviruses. I believe many of the central cell line repositories store cell lines in glass vials as well.

I'd be interested in the authors' thoughts.

Time to brush differences aside

Udaykumar Ranga — 2009-05-28T00:00:00Z

Recently I was asked to write an article on HIV Nobel prize for an Indian journal 'Resonance' that has wide circulation among colleges and universities. In this connection, I had to do quite a bit of reading digging up a lot of information from every possible source. The task proved to be much more difficult than I thought for three important reasons. First, the article was intended for the young students of biology therefore must be written in a simple language. Second, most of the information available was too biased often laced with personal prejudices therefore making it difficult to see which was genuine and which was not. Third, the viral contamination matters were quite confusing and no available information offered comprehensive idea on this issue. It took nearly a month for me to place the issues in the right perspective and a period of three more months to write the article. The article is freely accessible at the following URL: http://www.ias.ac.in/resonance/May2009/p472-498.pdf.

Having acquainted reasonably well with the history of HIV discovery and armed with clearer understanding on the viral contamination matters, I felt quite enthusiastic to see the article by Anders Vahlne in 'Retrovirology'. I found the article quite skillfully written and I must admit that I learnt a little more on the history of HIV discovery. However, I was quite disappointed to see that this article appears to have a predetermined agenda rather than adapting a balanced strategy. The article brilliantly portrayed the valuable contributions made by Gallo towards the science of HIV-1. The article, nevertheless, appears to have ignored the fact that the significance of Gallo’s scientific contributions has never been in dispute. Having read countless number of blogs, commentaries, perspectives, official documents etc, it is evident that even people who disliked Gallo’s personality did not deny the importance of his work. Like many other previous pieces of work that took up on themselves to defend Gallo, Vahlne’s article too appears to have suffered from similar kind of limitations. On two important matters, as discussed below, Vahlne’s article maintained silence and ignored facts.

Did Gallo attempt to misappropriate credit from others? Historically, all the disputes of HIV discovery have been centered around this single question, not on the merits of Gallo’s contributions to science. Any article that attempts to review HIV discovery must answer this question and the one below. Vahlne’s article appears to refuge even to consider this question. The most incriminating evidence that Gallo attempted to steal credit of HIV discovery for himself comes from the press meeting on 23rd April 1984 at NIH. At that meet, not only Margaret Heckler, secretary of the Department of Health and Human Services (DHHS), but also Gallo himself officially declared that AIDS virus was discovered in his own laboratory and it was named HTLV-III. In the entire press conference, or in his scientific presentation, Gallo did not make a mention to Montagnier. Do not forget that it was only a few days before this press meet that Gallo was in Montagnier’s laboratory and both of these teams have had an understanding of organizing a joint press meeting, involving both of the French and American groups. Importantly, Gallo was fully aware of the contributions made by the French group at the time of the press conference. Additionally, there was even evidence that the photos of the virus used by Gallo in the press meet actually belonged to French data. Investigation committees later identified that the virus photos shown at the conference were actually taken from the French team without their knowledge. It is practically impossible now for anyone to verify the veracity of such allegations for numerous reasons. Furthermore, the French group had already published their findings a year ago whereas Gallo’s publications were yet to appear a week later. Unfortunately Vahlne’s article maintains a stoic silence on this important evidence.

Why was the French application for HIV-ELISA delayed and eventually rejected by the US patents Office? Montagnier and his coworkers applied for a patent of the United Kingdom on 15th September 1983 and for a US patent on 5th December 1983. This application was kept pending at the US patent office for years before finally rejecting it. Five months later, on 23rd April 1984, Gallo and Popovic applied for a US patent for a diagnostic kit for AIDS. A year later on 28th May, 1984, the US Patent and Trademark Office awarded an American patent to Gallo and DHHS. The French application which was filed 17 months earlier was still kept pending in clear violation of the patent norms. While the facts remain such, it is quite surprising that the Vahlne’s article openly defends that ‘the approval of the Pasteur patent was delayed, principally because the French had not reduced their patent to practice, i.e. showed that they had a working blood test in the patent application’. Polarized statements like this raise doubts in readers’ mind as per the balanced approach of this article. In fact a lot of money was at stake. The patent would be worth hundreds of millions of Dollars. Several companies already applied for licenses to develop a commercial kit from this technology. If not for the commercial interests, is it not amazing that big laboratories should make serious claims on a simple technique like ELISA which any ordinary laboratory with basic knowledge in immunology should be able to develop?

Did Gallo’s publications establish the causative relationship between HIV and AIDS? Like many other articles have done previously, Vahlne’s article too presumes that Gallo’s publications ‘for the first time convincingly demonstrated that AIDS was caused by a new human retrovirus…’. Looking at various works of literature carefully, one would realize that a myth has been built around Gallo over the years. ‘The general understanding’ today is that while Montagnier was first to isolate the AIDS virus, it was Gallo who ‘accomplished’ to establish the cause and effect relationship between the virus and AIDS. The statements reinforcing such a notion are far from the truth. The single publication of Montagnier in 1983 and all the publications of Gallo in 1984 collectively did NOT establish that AIDS was caused by their viruses. All these publications only demonstrated a significant association, but not a causative relationship, between AIDS and HIV. In fact, way back in 1984, neither of these groups even knew that they were fumbling with the same virus. Neither the French team nor the American group through their early publications fulfilled the Koch’s postulations to prove that HIV caused AIDS. The main difference between the publications of the French and American groups remains in the numbers of virus isolates and quantum of data. Unlike the French team, whose work was based on only two viral strains, the American team used many more viral strains. Additionally, the American team also did an analysis at a much broader level. However, it would be too generous to give the credit of establishing the causative relationship to the American group based on numbers.

According to Koch postulates, (1) a microorganism must be found in the body of the host suffering from the disease (2) the microorganism must be isolated from the host and grown in pure culture (3) the cultured microorganism should cause disease when introduced into a healthy host and (4) the microorganism must be reisolated from the experimental host and identified as being identical to the original microorganism. From the above it is clear that both of the French and American groups successfully accomplished the objectives 1 and 2, but not 3 and 4 of the Koch’s postulates. All of us are fully aware why satisfying conditions 3 and 4 of Koch’s postulates is not possible for practical reasons. The objective of establishing the causative relationship between HIV and AIDS has been fulfilled in the subsequent years by the contributions of numerous laboratories spread all over the world and mainly because of unfortunate accidental injuries.

After the rightful decision by the Nobel Committee to honor Luc Montagnier and Francoise Barre Sinoussi for HIV discovery, many believed that the disputes on this topic would come to a halt. The virus is still out there ravaging continents and killing millions of hapless people. Isn’t it time that we brush all the differences aside and invest our energies in moving forward?

Nobel Prize 2008 and Gallo

Amiya Sarkar — 2008-11-11T00:00:00Z

It is very unfortunate that the Nobel Prize community did not give Gallo his due credit for his enormous contribution in the field of AIDS research. Omission or commission;I, like many others, was disappointed. It not only gives fame, but more importantly, it inspires the person towards achieving more. I am sure Gallo will maintain his momentum, notwithstanding.

Article website inaccessible

Gokhan ERTAYLAN — 2008-11-10T00:00:00Z

The article website "http://bioafrica.mrc.ac.za/proteomics/index.html" is inaccesible.