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About viruses and viral disease

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Poliovirus vaccine, SV40, and human cancer

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SV40Deep sequencing – which identified a viral contaminant of the rotavirus vaccine Rotarix - could have revealed the presence of simian virus 40 (SV40) in the poliovirus vaccine, had the technique been available in the 1950s. Exposure of over 100 million Americans to SV40, and many more worldwide, could have been avoided, as well as the debate about the role of this monkey virus in human cancer.

SV40 was discovered by Maurice Hilleman in 1960 as a contaminant of poliovirus vaccine. It was present in batches of both the Salk and Sabin poliovirus vaccines produced and distributed from 1954 to 1963. The source was the rhesus and cynomolgous monkey kidney cells used to produce the vaccine. Even more troubling was the observation that SV40 could cause tumors in hamsters. By 1963 screening procedures were instituted to ensure the absence of SV40 in poliovirus vaccines. Ironically, monkey cells were used for poliovirus vaccine production because it was feared that human cells might contain unknown human cancer viruses.

SV40 does not cause tumors in its natural host – monkeys – because it kills infected cells. However, in the wrong host- such as a hamster – the viral replication cycle is incomplete and virions are not produced. At a very low frequency, pieces of the viral DNA become integrated into the host chromosomal DNA. Problems arise if these viral DNA fragments encode the viral T (tumor) antigen. This protein is essential for lytic replication (which takes place in monkey cells) because it kick-starts cellular DNA synthesis. The cellular DNA synthetic machinery is then co-opted for replication of the viral DNA. When only T antigen is present, the cells divide without stopping – they are transformed, and on the way to becoming a tumor. SV40 does not need to cause tumors as part of its life cycle; they are an aberrant result of having T antigen push the cells to divide. SV40 T antigen can transform human cells, and therefore in theory the virus could cause human tumors.

The results of epidemiological studies initiated in the 1960s through the 1970s, in which thousands of poliovirus vaccine recipients were studied, indicated that this population did not have an increased risk of developing cancer. More recent reports that SV40 viral DNA is present in human tumors have led to a debate on the contribution of this virus to human cancer. Some of the arguments for and against presence of SV40 in human cancers are presented below.

Evidence that SV40 is present in human tumors

  • SV40 DNA has been detected in several human tumors, including osteosarcoma, mesothelioma, and non-Hodgkin’s lymphoma. Similar tumors are induced by the virus in hamsters.
  • Poliovirus vaccine produced in 1954 contained a variant of SV40 that can be distinguished from common laboratory strains. This viral variant has been found in three non-Hodgkin’s lymphoma patients

Evidence that SV40 is not present in human tumors

  • SV40 DNA is not present in all samples of a cancer, and in some studies of mesotheliomas, it has not been detected in any.
  • SV40 viral DNA has been detected in tumors of those who could not have received contaminated poliovirus vaccine.
  • In a comparison of mesotheliomas and normal tissues, SV40 DNA has been detected as frequently in both.
  • Analysis of the SV40 sequences in mesotheliomas showed that the viral DNA was derived from a laboratory strain which contains a gap that is not present in the wild type viral genome.

Even if SV40 DNA were definitively shown to be present in human tumors, this would not answer the question of whether the virus caused the cancer. The debate on the role of SV40 in human malignancy illustrates the difficulty in establishing cause and effect, and provides ample impetus for using genomic technologies to ensure that vaccines and other biological products are free of adventitious agents.

Garcea, R., & Imperiale, M. (2003). Simian Virus 40 Infection of Humans Journal of Virology, 77 (9), 5039-5045 DOI: 10.1128/JVI.77.9.5039-5045.2003

López-Ríos F, Illei PB, Rusch V, & Ladanyi M (2004). Evidence against a role for SV40 infection in human mesotheliomas and high risk of false-positive PCR results owing to presence of SV40 sequences in common laboratory plasmids. Lancet, 364 (9440), 1157-66 PMID: 15451223

PEDEN, K. (2008). Recovery of strains of the polyomavirus SV40 from rhesus monkey kidney cells dating from the 1950s to the early 1960s Virology, 370 (1), 63-76 DOI: 10.1016/j.virol.2007.06.045

The Immortal Life of Henrietta Lacks

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immortal_lifeShortly after I wrote about my years of experience with HeLa cells, I was contacted by author Rebecca Skloot. One of her many questions was how I knew that I had produced 800 billion HeLa cells in my laboratory over 26 years. I learned that she was writing a book about Henrietta Lacks, whose tumor was the source of HeLa cells in 1951. Subsequently I had the privilege of reading an early draft of her book, The Immortal Life of Henrietta Lacks, which will be published next month.

I thought I knew enough about HeLa cells and their origins, but Rebecca’s book shattered that impression. I’ve worked with the cells all my career and have always appreciated them, and the fact that Henrietta gave science something fabulous, but the back story I didn’t appreciate. How the whole affair deeply affected that family, and what they went through. I want to thank Rebecca for working so hard to get the whole story. And for being nice enough that the family trusted her! She not only vividly portrays what the family went through, but shows what HeLa has meant to science, how unscrupulous people always want to take advantage of others, and the good and bad about science. In the end, I keep coming back to the same question: if we had informed consent laws back then, would Henrietta have said no? If so, it would have been a tremendous loss for science and medicine. Or should I say setback – because eventually there would have been others. That’s how science is: someone always makes the discovery, sooner or later.

There will be a public launch of the book on 1 February at 7pm at McNally Jackson Bookstore in New York City. Rebecca will read a bit from the book, talk about it, sign it, and answer questions. Below are the details of the public event. If you are in the New York area, and have an interest in science, I encourage you to attend. I will certainly be there!

Public Launch Event: Rebecca Skloot Discusses Her New Book “The Immortal Life of Henrietta Lacks”

Award winning science writer Rebecca Skloot discusses and signs her new book, The Immortal Life of Henrietta Lacks. Books available for sale at this launch event one day before the book’s official publication date. Free & open to the public.

Book description: Her name was Henrietta Lacks, but scientists know her as HeLa. She was a poor Southern tobacco farmer who worked the same land as her slave ancestors, yet her cells — taken without her knowledge — became one of the most important tools in medicine. The first immortal human cells grown in culture, they are still alive today, though she has been dead for more than sixty years. If you could pile all HeLa cells ever grown onto a scale, they’d weigh more than 50 million metric tons — more than 100 Empire State Buildings. HeLa cells were vital for developing the polio vaccine; uncovered secrets of cancer, viruses, & the effects of the atom bomb; helped lead to important advances like in vitro fertilization, cloning, and gene mapping; and have been bought and sold by the billions. Yet Henrietta Lacks remains virtually unknown, buried in an unmarked grave. Now Rebecca Skloot takes us on an extraordinary journey, from the colored ward of Johns Hopkins Hospital in the 1950s to stark white laboratories with freezers full of HeLa cells; from Henriettas small, dying hometown of Clover, Virginia — a land of wooden slave quarters, faith healings, and voodoo — to East Baltimore today, where her children and grandchildren live, and struggle with the legacy of her cells. Henriettas family did not learn of her immortality until more than twenty years after her death, when scientists began using her husband and children in research without informed consent. The story of the Lacks family — past and present — is inextricably connected to the dark history of experimentation on African Americans, the birth of bioethics, and the legal battles over whether we control the stuff we are made of. More information at rebeccaskloot.com.

“Skloot’s book is wonderful, deeply felt, gracefully written, sharply reported.” — Susan Orlean, author of The Orchid Thief

“This is an extraordinary book, haunting and beautifully told.” — ERIC SCHLOSSER, author of Fast Food Nation

When: Monday, February 1 2010, 07:00 PM
Where: McNally Jackson Books, 52 Prince Street, New York, NY

Merkel cell polyomavirus, a new oncogenic human virus?

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merkel-cell-polyomavirusNone of the four human polymaviruses that were known in early 2008 – JC, BK, KI and WU – had been shown to cause cancer. The subsequent identification of a new polyomavirus associated with Merkel cell carcinoma demonstrates the type of evidence that is required to prove that a virus is oncogenic in humans.

Merkel cell carcinoma (MCC) is a relatively rare human skin cancer, although its incidence has increased in the past twenty years from 500 to 1500 cases per year. This cancer occurs more frequently than expected in individuals who are immunosuppressed, such as those who have received organ transplants or who have AIDS. A similar pattern of susceptibility is also observed for Kaposi’s sarcoma, a tumor that is caused by the herpesvirus HHV-8. Therefore it was suggested that MCC might also be caused by an infectious agent.

To identify the etiologic agent of MCC, the nucleotide sequence of total mRNA from several MCC tumors was determined and compared with the sequence of mRNA from a normal human cell. This analysis revealed that the MCC tumors contained a previously unknown polyomavirus which the authors named Merkel cell polyomavirus (MCV or MCPyV). The viral genome was found to be integrated at different sites in human chromosomal DNA from MCC tumors.

If MCV infection causes MCC, then the viral genome should be present in tumors but not in normal tissues. MCC DNA was found in eight of ten MCC tumors, each obtained from a different patient. The viral genome was not detected in various tissues samples from 59 patients without MCC. Furthermore, the viral genome had integrated into one site in the chromosome of one tumor and a metastasis dervied from it. This observation indicates that integration of the viral genome occurs first, before division of the tumor cells.

In subsequent studies MCV DNA has been detected in 40-85% of the MCC tumors examined. The viral DNA is not found in small cell lung carcinoma, which, like MCC, is also a neuroendocrine carcinoma. MCV particles have also been detected by electron microscopy in the cytoplasm and nucleus of tumor cells from one patient, suggesting ongoing viral replication.

How might MCV cause Merkel cell carcinoma? Expression of the viral protein known as T antigen might be sufficient to transform cells. Alternatively, integration of the viral DNA into human DNA could lead to unregulated synthesis of a protein that transforms cells. To prove that MCV causes Merkel cell carcinoma, it will be necessary to demonstrate that infection with the virus, or transfection with viral DNA, transforms and immortalizes cells in culture.

Feng, H., Shuda, M., Chang, Y., & Moore, P. (2008). Clonal Integration of a Polyomavirus in Human Merkel Cell Carcinoma Science, 319 (5866), 1096-1100 DOI: 10.1126/science.1152586

Wetzels, C., Hoefnagel, J., Bakkers, J., Dijkman, H., Blokx, W., & Melchers, W. (2009). Ultrastructural Proof of Polyomavirus in Merkel Cell Carcinoma Tumour Cells and Its Absence in Small Cell Carcinoma of the Lung PLoS ONE, 4 (3) DOI: 10.1371/journal.pone.0004958

Prevalence of human polyomaviruses

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murine-polyomavirusWhile immunosuppressive therapy can ameliorate a variety of diseases, one unfortunate side effect of the treatment is that it may lead to pathogenic infections by viruses which would otherwise be benign. An example is the brain infection PML which occurs when immunosuppression leads to replication of the polyomavirus JC. How many polyomaviruses do we have to worry about, and how frequently do they infect humans?

There are five known human polyomaviruses: JC and BK, both isolated in 1971, and the more recently discovered KIV, WUV, and MCV (Merkel cell polyomavirus). The percentage of the human population that is infected with these viruses has been addressed by determining whether antibodies directed against the viral capsid protein VP1 are present in sera.

A total of 1501 serum specimens from healthy blood donors were examined. The presence of antibody to the viruses – known as seroprevalence – was 82% for BKV, 39% for JCV, 15% for LPV, 55% for KIV, 69% for WUV, and 25 and 42% for two different MCV strains. The sampling error is plus or minus 1%. Seroprevalence of these viruses was also examined in samples from 721 children, and the results were similar to those obtained with adult sera. From this information the authors conclude that polyomavirus infection of humans probably occurs during childhood.

The authors also determined the level of antibodies to a monkey polyomavirus, SV40. This virus was introduced into millions of people as a contaminant of early preparations of inactivated and infectious poliovirus vaccines. Whether or not the virus causes tumors in humans is a matter of scientific and legal controversy. Only 2% of the specimens were positive for antibodies to SV40. The low seroprevalence of antibodies to SV40 suggest that this virus does not circulate extensively among humans.

Given the high seroprevalence of polyomaviruses in humans, it is not surprising that they are significant pathogens in immunosuppressed populations. An important question is why these viruses can peacefully co-exist in many humans without causing disease. Are human polyomaviruses simply passengers, or do they benefit us in some unknown way?

Kean, J., Rao, S., Wang, M., & Garcea, R. (2009). Seroepidemiology of Human Polyomaviruses PLoS Pathogens, 5 (3) DOI: 10.1371/journal.ppat.1000363