TWiV 158: Wolverines go viral

poliovirus + CD155Hosts: Vincent RacanielloRich Condit, Alice Telesnitsky, and Kathy Spindler

Vincent and Rich visit the Microbiology and Immunology Department at the University of Michigan Medical School, and speak with Alice and Kathy about their work on HIV genome dimerization, and packaging and pathogenesis of mouse adenovirus.

Model of poliovirus bound to CD155 made by Stefan Taube.

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Alice – Banvard’s Folly by Paul S. Collins
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TimDead Ends to Somewhere by Richard L. Ward
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TWiV 157: Better innate than never

dendritic cellHosts: Vincent RacanielloRich Condit, Alan Dove, Dickson Despommier, Jeremy Luban, and Gabriel Victora

A large TWiV panel remembers Ralph Steinman, and considers a new innate sensor of retroviral capsids.

Photograph of a dendritic cell (green) interacting with T cells (cyan) near a blood vessel by Gabriel Victora.

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Dickson – Eats, Shoots & Leaves by Lynne Truss
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AlanColeman LED quad lantern
GabrielA History of Immunology by Arthur M. Silverstein
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Vincent – Principles of Molecular Virology by AJ Cann

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DavidCarl Sagan’s Pale Blue Dot (YouTube)

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TWiV 154: Symbiotic safecrackers

mmtvHosts: Vincent Racaniello, Alan Dove, and Rich Condit

Vincent, Alan, and Rich are very enthusiastic about two studies that show how gut bacteria help viral invaders.

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Alan – SciWriteLabs
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TonyIntroduction to AI online course

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Trust science, not scientists

XMRVWhether or not the retrovirus XMRV is a human pathogen has been debated since the virus was first described in 2006. The answer is now clear: the results of Blood XMRV Scientific Research Group, along with a partial retraction of the 2009 Science paper describing identification of the retrovirus in patients with chronic fatigue syndrome (CFS) show that detection of XMRV in patient samples is a result of contamination.

The Blood XMRV group obtained new blood samples from 15 individuals previously shown to be positive for XMRV (Lombardi et al., 2009) or MLV (Lo et al., 2010) ; 14 of these were from CFS patients. Fifteen blood samples were also obtained from healthy donors. The samples were coded and sent to 9 laboratories for analysis. These laboratories (Abbott Molecular, Abbott Diagnostics, CDC, FDA/Lo, FDA/Hewlett, Gen-Probe, NCI/DRP, and WPI) conducted validated assays for viral nucleic acid, viral replication, or viral antibodies. Positive control samples were also included that were ‘spiked’ with XMRV, in the form of cell culture fluids from the cell line 22Rv1. Each laboratory was at liberty to choose which assays to carry out.

Two laboratories reported evidence of XMRV in the coded samples.  Only WPI identified positive specimens by PCR: two from negative controls, and one from a CFS patient. The FDA/Lo laboratory did not detect any positives by PCR, using the same nested assay that they had previously reported in their published study. The samples tested included 5 specimens that were positive in the Lo et al. study.

Lombardi and colleagues have previously concluded that viral culture is the most sensitive method for detecting XMRV; however the FDA/Hewlett laboratory failed to culture virus from CFS samples. This laboratory did culture virus from positive control specimens, demonstrating the sensitivity of their methods. The FDA/Ruscetti laboratory recovered virus from 3/15 CFS samples but also from 6/15 negative control specimens. WPI did not carry out viral culture assays due to contamination of their cell lines with mycoplasma.

Four laboratories tested the samples for the presence of antibodies that react with XMRV proteins. Only WPI and NCI/Ruscetti detected reactive antibodies, both in CFS specimens and negative controls. There was no statistically significant difference in the rates of positivity between the positive and negative controls, nor in the identity of the positive samples between the two laboratories.

These results demonstrate that XMRV or antibodies to the virus are not present in clinical specimens. Detection of XMRV nucleic acid by WPI is likely a consequence of contamination. The positive serology reported by WPI and NCI/Ruscetti laboratories remained unexplained, but are most likely the result of the presence of cross-reactive epitopes. The authors of the study conclude that ‘routine blood screening for XMRV/P-MLV is not warranted at this time’.

One of the authors on Lombardi et al., Robert Silverman, decided to reexamine some of the DNA preparations from CFS patients that were originally used to detect XMRV DNA by PCR. He found that all the positive specimens from CFS patients were contaminated with XMRV plasmid DNA. Therefore the authors of the original study have retracted Figure 1 (single-round PCR detection of XMRV in CFS PBMC DNA); table S1, XMRV sequences, and figure S2, phylogenetic analysis of XMRV sequences.

A puzzling aspect of Silverman’s results is that XMRV plasmid DNA was detected only in samples from CFS patients, not healthy controls. This pattern would not be expected if the specimens were properly blinded, that is, coded so that the investigators did not know which were controls and which were from CFS patients. The authors offer no explanation of these findings.

The paper reporting contamination of samples with XMRV is entitled ‘Partial Retraction‘. It’s not clear to me why the entire paper has not been retracted. After removing the PCR and nucleic acid sequencing results, there is no evidence indicating the presence of XMRV in the patient samples. The remaining experiments show detection of a retrovirus by cell culture experiments, and the presence of viral proteins or antibodies to the virus in clinical specimens. None of these findings prove that what is being studied is XMRV. The title of the original paper ‘Detection of an infectious retrovirus’, XMRV, in blood cells of patients with chronic fatigue syndrome‘, is unsupported.

In an accompanying article on the XMRV story entitled ‘False Positive‘, Judy Mikovits of WPI notes that “Anyone who says this is a lab contaminant has drawn the wrong conclusion and has done a disservice to the public”. She goes on to imply that a gammaretrovirus is likely involved in CFS. On the contrary, pursuing the CFS-gammaretrovirus hypothesis is a disservice to those with CFS, and detracts from efforts to solve the disease. There are no data to support such an association, and to suggest that a lab contaminant, XMRV, has pointed the way to a bona fide etiologic agent seems implausible.

XMRV does not cause CFS. The virus arose in mice between 1993-96, and its detection in patient samples is clearly a result of contamination. Reaching these conclusions has required a long and often contentious journey that has highlighted the best and worst aspects of scientific research. There are many lessons to be learned from XMRV, but an important one is that science progresses not from the work of a single investigator, but from the collective efforts of many laboratories. XMRV reminds us to trust science, not scientists.

TWiV 143: Live at ASV in Minneapolis

asv minneapolisHosts: Vincent Racaniello, Rich Condit, Julie Overbaugh, and Stacey Schultz-Cherry

Vincent, Rich, Julie and Stacey recorded TWiV at the 30th Annual Meeting of the American Society for Virology in Minneapolis, where they discussed the role of neutralizing antibodies in protection against HIV-1 infection, and astroviruses, agents of gastroenteritis.

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Rich – Iter – building a fusion reactor
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JingBees by Rudolph Steiner

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TWiV 136: Exit XMRV

nude mouseHosts: Vincent Racaniello, Alan Dove, Rich Condit, and Stephen Goff

Retrovirologist Stephen Goff joins Vincent, Rich, and Alan for a discussion of recent papers on the retrovirus XMRV and its association with chronic fatigue syndrome and prostate cancer.

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Alan – The Demon-Haunted World by Carl Sagan
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– Trends in annual rates of death (pdf)
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Raihan  – Bruce Aylward: How we’ll stop polio for good (YouTube)

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XMRV is a recombinant virus from mice

recombinant xmrvThe novel human retrovirus XMRV has been associated with prostate cancer and chronic fatigue syndrome. The nucleotide sequence of XMRV isolated from humans indicates that the virus is nearly identical with XMRV produced from a human prostate tumor cell line called 22Rv1. This cell line was derived by passage of human prostate tumor tissue in nude mice. Sequence analyses reveal that the genomes of these mouse strains contain two different proviral DNAs related to XMRV. These viral genomes recombined to produce XMRV that has been isolated from humans.

XMRV was originally isolated from a human prostate cancer in 2006, and subsequently associated with ME/CFS. The human cell line 22Rv1, which was established from a human prostate tumor (CWR22), produces infectious XMRV. An important question is whether XMRV was present in the original prostate tumor, or was obtained by passage through nude mice. To answer this question, DNA from various passages of the prostate tumor in nude mice (called xenografts), and the mouse strains used to passage the tumor, were analyzed for the presence of XMRV proviral DNA.

Early-passage xenografts did not contain XMRV, but mouse cells found in them did contain two related proviruses called PreXMRV-1 and PreXMRV-2. The 3’-3211 nucleotides of PreXMRV-1, and both LTRs, are identical to XMRV save for two nucleotide differences. The genomic 5’-half of XMRV and PreXMRV-1 differs by 9-10%. PreXMRV-1 is defective for replication due to mutations in genes encoding the gag and pol proteins. PreXMRV-2 does not contain obvious mutations that would prevent the production of infectious viruses. The gag-pro-pol and a part of the env region of this viral genome is identical to that of XMRV save for two base differences; the LTRs and the remainder of the genome differ by 6-12% from XMRV.

Comparison of the sequences of PreXMRV-1 and PreXMRV-2 indicates that recombination between the two viral genomes led to the formation of XMRV. When the sequences of PreXMRV-1 and −2 are used to construct the recombinant XMRV, the resulting virus differs by only 4 nucleotides from the consensus XMRV sequence derived from all human isolates reported to date.

The nude mice used for passage of the original prostate tumor were likely the NU/NU and Hsd strains. Neither mouse strain contains XMRV proviral DNA, but both contain PreXMRV-1 and PreXMRV-2 proviral DNA.

These data demonstrate that XMRV was not present in the original CWR22 prostate tumor, but arose by recombination of PreXMRV-1 and PreXMRV-2 between 1993-1996. When the original prostate tumor was implanted into nude mice, some of the mice harbored both pre-XMRV-1 and −2 endogenous proviruses, which recombined to form XMRV. The authors believe that XMRV originating from the CRWR22 xenografts, the22Rv1 cell line, or other related cell lines has contaminated all human samples positive for the virus. In addition, they suggest that PCR assays for XMRV may actually detect PreXMRV-1 and −2 or other endogenous viral DNA from contaminating mouse DNA.

Another possibility to explain the origin of XMRV is that it arose in mice and can infect humans. If this is true, then XMRV would have to be present in the nude mice used to passage the CWR22 human prostate tumor. No evidence for an XMRV provirus was found in 12 different nude mouse strains, including two used to passage the CWR22 tumor. Furthermore, a screen of 89 inbred and wild mice failed to reveal the presence of proviral XMRV DNA. Hence the authors conclude:

…that XMRV arose from a recombination event between two endogenous MLVs that took place around 1993-1996 in a nude mouse carrying the CWR22 PC xenograft, and that all of the XMRV isolates reported to date are descended from this one event.

It is possible that XMRV produced during passage of CWR22 in nude mice subsequently infected humans. Because XMRV arose between 1993-1996, this scenario could not explain cases of prostate cancer and chronic fatigue syndrome that arose prior to that date.

How can these findings be reconciled with the published evidence that sera of ME/CFS patients from the 1980s contain antibodies to XMRV? Those antibodies were not shown to be directed specifically against XMRV, and therefore cannot be used to prove that XMRV circulated in humans prior to 1993-96. Furthermore, in the absence of clear isolation of an infectious virus, antibody tests alone have proven highly unreliable for identification of new viruses.

Where do these findings leave the hypothesis that XMRV is the etiologic agent of prostate cancer and ME/CFS? All published sequences of human XMRV isolates are clearly derived by recombination of PreXMRV-1 and −2. The finding of human XMRV isolates that are not derived from PreXMRV-1 and −2 would leave a role for XMRV in human disease. As of this writing, no such XMRV isolates have been reported in the scientific literature.

Update: A second paper has also been published in Science Express today entitled “No evidence of murine-like gammaretroviruses in CFS patients previously identified as XMRV-infected”. Editors of the journal Science have asked the authors to retract their 2009 paper linking XMRV infection with chronic fatigue syndrome. The authors have refused.

T. Paprotka, K. A. Delviks-Frankenberry, O. Cingoz, A. Martinez, H.-J. Kung, C.G. Tepper, W-S Hu, M. J. Fivash, J.M. Coffin, & V.K. Pathak (2011). Recombinant origin of the retrovirus XMRV. Science Express

TWiV 135: Live in the Big Easy

ASM GM New OrleansHosts: Vincent Racaniello, Roger Hendrix, Rachel Katzenellenbogen, and Harmit Malik

Vincent and guests Rachel Katzenellenbogen, Roger Hendrix, and Harmit Malik recorded TWiV #135 live at the 2011 ASM General Meeting in New Orleans, where they discussed transformation and oncogenesis by human papillomaviruses, the amazing collection of bacteriophages on the planet, and the evolution of genetic conflict between virus and host.

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Syncytin knockout mice show role for endogenous retroviral gene (PNAS)
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Not-so-similar fate of identical twins infected with HIV-1

identical twinsFor extra credit in my recently concluded virology course, I asked students to summarize a virology finding in the style of this blog. I received many excellent submissions which I plan to post here in the coming months.

by Amanda Carpenter

In 1983, identical twin boys simultaneously received a contaminated blood transfusion immediately after birth, and were subsequently diagnosed with HIV-1. Years later, one of the twins is faring very well and has a near normal immune system, while the other is in poor health and has experienced many complications. How could the same virus, infecting two individuals at the same time, with the same genetic background, yield such different clinical courses? This unfortunate natural experiment has allowed researchers to study viral evolution while holding host genetic make-up constant. Brigham Young University Chairman of Biology Keith Crandall has studied the virus in this interesting case and recently published his findings in BMC Evolutionary Biology.

Crandall’s team examined blood samples obtained from the twins when they were 15 years old. They collected nucleotide sequence data from several genes essential to the success of HIV infection, which allowed them to extrapolate rates of viral growth, recombination, and genetic diversity. They reported that the virus of the healthier twin exhibited generally higher growth rates, higher genetic diversity, and higher recombination rates than the virus of the sicker twin (2).

How is this possible? Infected individuals produce an estimated 1010 new HIV virions every day, with errors occurring at a rate of about 1 per 104 nucleotides incorporated (1). These frequent point mutations are a simple starting point to explain the divergence of a once identical virus. In addition, HIV virions are capable of exchanging their genetic material with a different strain of virus via a process called recombination. Recombination is likely a random event, but has important implications for the host immune system.

Part of the immune system’s response to HIV is the utilization of cytotoxic T lymphocytes (CD8+ cells), which target and kill virus-infected cells. These cells are very specific, and if a recombination event occurs, these cytotoxic cells may not be able to recognize the new viral strain as readily as the original. The immune system may adapt to the new strain, but the virus may recombine again and again, and the immune system will not be able to keep up. These recombinant strains are likely to become more prevalent through natural selection. If recombined strains are better at evading the immune system, and are therefore more detrimental to the host, does this mean they are more successful? Why would the virus that has higher genetic diversity, a growth rate, and a higher recombination rate cause less disease? Perhaps the answer lies in the immune system.

Once out of the womb, these twins no longer exist in identical environments. They are exposed to different pathogens, bacteria, and microbes, all of which affect the make-up of the immune system. The healthier twin’s immune system may be better able to fight the virus, and so the virus must grow, diversify, and recombine in order to propagate the infection. In other words, because the sicker twin has a more depressed immune system, the virus is replicating with less resistance, and there is less incentive for the virus to evolve. Divergent viral evolution in the case of these monozygotic twins is likely due to random mutation and recombination events, combined with antiviral pressure from the hosts, whose immune systems are not identical at all.

(1) Yang, O., Church, J., Kitchen, C., Kilpatrick, R., Ali, A., Geng, Y., Killian, M., Sabado, R., Ng, H., Suen, J., Bryson, Y., Jamieson, B., & Krogstad, P. (2005). Genetic and Stochastic Influences on the Interaction of Human Immunodeficiency Virus Type 1 and Cytotoxic T Lymphocytes in Identical Twins Journal of Virology, 79 (24), 15368-15375 DOI: 10.1128/JVI.79.24.15368-15375.2005

(2) Tazi L, Imamichi H, Hirschfeld S, Metcalf JA, Orsega S, Pérez-Losada M, Posada D, Lane HC, & Crandall KA (2011). HIV-1 infected monozygotic twins: a tale of two outcomes. BMC evolutionary biology, 11 PMID: 21385447

TWiV 133: The HIV hideout

Dr. Kathleen CollinsHosts: Vincent Racaniello, Rich Condit, Dickson DespommierAlan Dove, and Kathleen Collins

Vincent, Rich, Alan, and Dickson discuss the cellular reservoir of HIV-1 with Kathleen Collins, MD, PhD.

Click the arrow above to play, or right-click to download TWiV #133 (42 MB .mp3, 87 minutes).

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Kathleen – TRIM5 is an innate immune sensor (Nature)
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Vincent – Magnet Balls (Amazon)

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Chris  – Periodic Tales by Hugh Aldersey-Williams

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