Murine gammaretroviruses in prostate cancer cell lines

nude mouseThe retrovirus XMRV arose during passage of a human prostate tumor in nude mice. The genomes of these mice contain two different proviral DNAs related to XMRV, pre-XMRV-1 and pre-XMRV-2, that recombined to produce XMRV that has been isolated from humans. Two other prostate cancer cell lines also contain mouse gammaretroviruses that are not XMRV. These viruses may have entered the prostate tumor cells during xenograft passage in immunocompromised mice.

The discovery of infectious XMRV in the prostate tumor cell line 22Rv1 prompted the examination of other prostate tumor cell lines for the presence of murine gammaretroviruses. Antisera against Moloney murine leukemia virus were used to screen 72 cell lines by immunohistochemistry for the presence of murine gammaretroviruses. Three human prostate tumor cell lines (22Rv1, LAPC4, and VCaP) and one lung carcinoma cell line (EKVX) reacted with the antisera.

Polymerase chain reaction and nucleotide sequencing analysis revealed that these viruses are not XMRV. The virus in the EKVX cell line is a xenotropic MLV similar to a virus previously isolated from a human B-lymphoblastoid cell line. The virus from the LAPC4 and VCaP cell lines is the murine xenotropic retrovirus Bxv-1. A different sample of VCaP cells obtained from the ATCC were also positive for Bxv-1, as were LAPC4 cells obtained from a different laboratory. Replication-competent viruses were detected in all three cell lines.

How did these human prostate cancer cell lines become contaminated with murine gammaretroviruses? The authors believe this is a consequence of passage of the tumors through immunocompromised mice. In support of this hypothesis, the retrovirus Bxv-1 is present in nude mice, and passage of tumors in these mice can lead to infection with xenotropic MLVs. In contrast to the origin of XMRV, recombination was not needed to produce these viruses.

These findings led the authors to examine two other prostate cancer cell lines, present in their laboratory, DU145 and LNCaP, for the presence of gammaretroviruses. They found these cell lines to be contaminated with XMRV, likely obtained from 22RV1 cells in use in the laboratory. Fresh aliquots of the DU145 and LNCaP cells obtained from other sources were not contaminated. The authors conclude that if

CWR22Rv1 cells are routinely cultured in a typical biomedical research laboratory setting (e.g. using standard Class II biosafety cabinets and procedures for cell culture in which two different cell lines are never present under the hood at the same time), that XMRV can infect and contaminate other cell lines.

Karen Sandell Sfanos, Amanda L. Aloia, Jessica L. Hicks, David M. Esopi, Jared P. Steranka, Wei Shao, Silvia Sanchez-Martinez, Srinivasan Yegnasubramanian, Kathleen H. Burns, Alan Rein, & Angelo M. De Marzo (2011). Identification of Replication Competent Murine Gammaretroviruses in Commonly Used Prostate Cancer Cell Lines PLoS One : 10.1371/journal.pone.0020874

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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XMRV infection of Rhesus macaques

rhesus macaqueThe first detailed study of infection of nonhuman primates with the retrovirus XMRV reveals that the virus establishes a persistent infection characterized by infection of multiple tissues. Viremia (virus in the blood) is low and transient, with proviral DNA detectable in blood lymphocytes. The results show that the Rhesus macaque can be used to study XMRV infection, transmission, vaccines, and antiviral drugs.

The subject of this study, the Rhesus macaque (Macaca mulatta), was selected because of its evolutionary proximity to humans and a comparable immune system. The monkeys used did not have antibodies to the capsid protein p30 of XMRV, indicating that they were not previously infected. Animals were inoculated intravenously with 3.6 million TCID50 of purified XMRV – a good amount of virus, to ensure infection. The virus used, VP62, was produced by transfecting cells with cloned viral DNA isolated from human prostate.

Virus in the plasma fraction of blood was assayed by quantitative RT-PCR. Of three animals infected, virus was detected in one animal at day 4 and not after day 14; and in a second animal from days 14-20. The third animal did not develop detectable viremia. Proviral DNA was found in peripheral blood mononuclear cells (PBMC) of all three monkeys for 3-4 weeks, indicating successful infection. At one month post-infection proviral DNA was no longer detected. Plasma virus was again detected in one of the positive animals on day 291, 16 days after being immunized with a mixture of XMRV proteins. This means that viral DNA had been present in this animal but was not detected. XMRV was detected in CD4+ and CD8+ T cells and NK cells, but not in B cells or monocytes.

Rhesus macaques infected with XMRV did not display obvious clinical symptoms. Analysis of peripheral blood revealed increases in the number of circulating B and NK cells. Anti-viral antibody titers were detected after infection and re-infection of animals but soon decreased.

Other infected animals were sacrificed during the acute phase of infection to identify pathological changes and sites of virus replication. No pathogenic consequences were observed except for the formation of germinal centers in spleen and lymphoid organs, changes that are expected after immune stimulation. Virus was detected in a wide variety of tissues, including spleen, lymph nodes, the lining of the gastrointestinal tract, prostate, testis, cervix, vagina, and pancreas, but not* in others including brain, heart, kidney, and bladder. Different types of cells were infected in different tissues: lymphocytes in lymphoid organs, macrophages in lung, epithelial or interstitial cells in other organs. The authors note that “this viral behavior appears specific to this virus”.

Here are some other comments and conclusions drawn from this study:

  • The authors suggest that in Rhesus macaques, XMRV causes first an acute infection, followed by a persistent chronic infection. A persistent infection lasts for long periods of time; a chronic infection is a persistent infection that is eventually cleared. Since the monkeys in this study were all sacrificed, it’s not possible to determine if the infection was cleared.
  • The presence of XMRV in certain blood cells resembles the pattern in a cohort of ME/CFS patients
  • Virus is present in the prostate early in acute infection – XMRV was identified in prostate tumors
  • The presence of XMRV in reproductive tract tissues is consistent with sexual transmission of infection
  • After the acute phase, virus levels are very low, but there could be a different outcome in individuals with immune dysfunction
  • One animal produced virus after immunization; perhaps immune activation results in cycles of virus production
  • The virus has an initial acute phase followed by reactivation. The authors comment: “While our study has not linked XMRV infection with pathogenic mechanisms that might lead to prostate cancer or chronic fatigue syndrome, we submit that such link, assuming it exists, would be a temporally distant one.”
  • It would be informative to determine if XMRV is present in some of the same tissues in humans that were observed to be infected in rhesus macaques

Because the study involved only a small number of monkeys (8), the experiments should be repeated with additional animals, and in different laboratories, to verify the findings. I also wonder if the choice of the intravenous inoculation route had an effect on the pattern of infection and tropism. It is well known that viral pathogenesis can be determined by how the virus enters the host. For example, the same virus may replicate in different tissues, or have different virulence, when inoculated in different ways. This question can be readily addressed by inoculating rhesus macaques via different routes.

Studying viral pathogenesis (the series of events that occur during viral infection of a host) in animals is essential for understanding how viruses cause disease in humans. However, the results of such studies must always be interpreted with caution, because what is true in an animal is not always true for a human. For example, simple differences in size, metabolism, and development can have substantial effects on pathogenesis. In interpreting the results of animal studies, we must keep in mind the adage, ‘Mice lie, monkeys exaggerate‘.

Update: *These are the results of immunohistochemistry (IHC), which detects viral proteins and likely the produce of viral replication. When the IHC-negative tissues were examined for the presence of viral nucleic acids, low frequency signals were detected. The authors speculate that this is likely a consequence of failure of XMRV to replicate in these tissues.

Onlamoon, N, DasGupta, J, Sharma, P, Rogers, K, Suppiah, S, Rhea, J, Molinaro, RJ, Gaughan, C, Dong, B, Klein, E, Qui, X, Devare, S, Schochetman, G, Hackett, J, Silverman, R, & Villinger, F (2011). Infection, viral dissemination and antibody responses of Rhesus macaques exposed to the human gammaretrovirus XMRV Journal of Virology