Viral RNA is not infectious virus!

Zika RNA and virusA study of sexual transmission of Zika virus among mice (link to paper) demonstrates beautifully that viral nucleic acid detected by polymerase chain reaction (PCR) is not the same as infectious virus.

Male mice were infected with Zika virus and then mated with female mice. Efficient sexual transmission of the virus from males to females was observed. This observation in itself is very interesting but is not the focus of  my comments.

To understand the dynamics of sexual transmission, the authors measured Zika virus shedding in seminal fluid – by both PCR, to detect viral RNA, and by plaque assay, to detect infectious virus. The results are surprising (see figure – drawn in my hotel room).

Zika virus RNA persisted in semen for up to 60 days – far longer than did infectious virus, which could not be detected after about three weeks.

Many laboratories choose to assay the presence of viral genomes by PCR. This is an acceptable technique as long as the limitations are understood – it detects nucleic acids, not infectious virus.

Despite the presence of Zika virus RNA in seminal fluid for at least 60 days after infection, these mice are not likely to transmit virus after a few weeks. There is a lower limit of detection of the plaque assay – approximately 10 plaque forming units/ml – whether that would be sufficient to transmit infection is a good question.

Why Zika viral RNA and not infectious virus would persist for so long is an important and unanswered question that should definitely be studied.

Recently many papers have been published which demonstrate that Zika virus and Ebolavirus can persist in a variety of human fluids for extended periods of time. These results have been interpreted with alarm, both by scientists and by science writers. However, in most cases the assays were done by PCR, not by plaque assay, and therefore we do not know if infectious virus is present. Viral RNA would not constitute a threat to transmission, while infectious virus would.

The lesson from this study is very clear – in novel experimental or epidemiological  studies it is important to prove that any viral nucleic acid detected by PCR is actually infectious virus. Failing to do so clouds the conclusions of the study.

There are few excuses for failing to measure viral infectivity by plaque assays. Please don’t tell me it’s too much work – that’s a poor excuse on which to base selection of an assay. Even if your virus doesn’t form plaques there are alternatives for measuring infectious virus.

If you are wondering how a plaque assay is done, check out my short video below.

TWiV 393: Lovers and livers

Possible sexual transmission of Zika virus, and a cell protein that allows hepatitis C virus replication in cell culture by enhancing vitamin E mediated protection against lipid peroxidation, are the subjects discussed by the TWiVerati on this week’s episode of the science show This Week in Virology.

You can find TWiV #393 at microbe.tv/twiv, or listen below.

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TWiV 377: Chicken with a side of Zika

TWiVOn episode #377 of the science show This Week in Virology, the TWiVniks review the past week’s findings on Zika virus and microcephaly, and reveal a chicken protein that provides insight on the restriction of transmission of avian influenza viruses to humans.

You can find TWiV #377 at microbe.tv/twiv, or you can listen below.

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TWiV 361: Zombie viruses on the loose

On episode #361 of the science show This Week in Virology, the TWiVsters discuss Frederick Novy’s return from retirement to recover a lost rat virus, and evidence for persistence of Ebolavirus in semen.

You can find TWiV #361 at www.microbe.tv/twiv.

TWiV 312: She sells B cells

On episode #312 of the science show This Week in Virology, the TWiVbolans discuss the finding that human noroviruses, major causes of gastroenteritis, can for the first time be propagated in B cell cultures, with the help of enteric bacteria.

You can find TWiV #312 at www.microbe.tv/twiv.

TWiV 309: Ebola email

On episode #309 of the science show This Week in Virology, the TWiVocytes answer questions about Ebola virus, including mode of transmission, quarantine, incubation period, immunity, and much more.

You can find TWiV #309 at www.microbe.tv/twiv.

XMRV not detected in seminal plasma

How XMRV, the new human retrovirus associated with prostate cancer and chronic fatigue syndrome, might be transmitted among humans is unknown. The finding that the virus can be detected in prostate cancer cells, and in prostatic secretions of men with prostate cancer suggests that it could be sexually transmitted. To address this question, the presence of XMRV in seminal plasma of men with HIV-1 was examined. Although the virus was not detected in 93 samples from 54 HIV-1 infected men, the study provides little information on possible transmission mechanisms of XMRV.

This study involved two groups of HIV-1 infected men from the Netherlands: 29 who have sex with men, and 25 heterosexual men. The rationale for examining HIV-1 infected men for XMRV was that “they have a higher chance of contracting sexually transmitted pathogens than non-HIV-1 infected men”. For 39 men a second sample was also available from another time point, bringing the total samples to 93.

To detect XMRV, semen samples were diluted 1:1 with buffer and centrifuged to remove cells, yielding seminal plasma. Total nucleic acid was then extracted and subjected to reverse-transcription and then polymerase chain reaction. This procedure assays for the presence of XMRV viral RNA. At the same time, the samples were also tested for the presence of HIV-1 RNA. The positive control for XMRV was total nucleic acid extracted from a prostate cancer cell line known to produce viral RNA.

The results show that HIV-1 was detected in 25% of the seminal plasma samples, while none contained XMRV nucleic acid. The authors conclude:

Although HIV-1 was amplified from 25% of the seminal plasma samples, no XMRV was detected, suggesting that either the prevalence of XMRV is very low in The Netherlands, or that XMRV is not naturally present in the seminal plasma.

In my opinion, these conclusions are not supported by the data obtained in this study. Here are my reasons:

  • The semen samples were subjected to centrifugation, which removes all cells, including spermatozoa, epithelial cells, and lymphocytes. Such cells could harbor virions.
  • The study was designed only to search for XMRV virions or viral RNA, not proviral DNA, which is integrated into cellular DNA.
  • No attempt was made to determine if the 54 men were infected with XMRV. This could have been done by taking blood samples and co-culturing them with LNCaP cells, then performing PCR. If none of the men were infected with the virus, then absence of the virus in their semen is meaningless.

To determine if XMRV could be transmitted in semen, I would obtain semen samples from patients known to be infected with the virus. Then I would co-culture total semen and seminal plasma with LNCaP cells to amplify any virus present, followed by PCR to detect either virions or proviral DNA. I realize it may be difficult to conduct the study in this way, but I don’t see the value of doing it any other way.

Marion Cornelissen, Fokla Zorgdrager, Petra Blom, Suzanne Jurriaans, Sjoerd Repping, Elisabeth van Leeuwen, Margreet Bakker, Ben Berkhout, & Antoinette C. van der Kuyl (2010). Lack of Detection of XMRV in Seminal Plasma from HIV-1 Infected Men in The Netherlands PLoS One : 10.1371/journal.pone.0012040

XMRV infection is enhanced by prostatic protein fragments

amyloid_temFragments of an abundant protein produced by the prostate form amyloid fibrils that enhance infection of cells by human immunodeficiency virus type 1. These fibrils, called semen-derived enhancer of virus infection (SEVI), have been found to boost infection of prostate cells by the retrovirus XMRV. Is this evidence that XMRV causes prostate cancer?

Because most HIV-1 infections are a consequence of genital exposure to semen of virus-infected men, seminal fluid was screened for peptides or proteins that enhance viral infectivity. Peptides (34 – 40 amino acids in length) derived from prostatic acidic phosphates, a common protein found in semen, were found to dramatically enhance HIV-1 infection of cultured cells. These peptide fragments form amyloid fibrils which bind both virions and cells, thereby promoting virus attachment and stimulating infectivity. The prostatic amyloid fibrils, called SEVI, are found at high levels in semen, which also stimulates HIV infection. SEVI is believed to play an important role in sexual transmission of HIV. Inhibitors of the stimulatory effect of SEVI on HIV infection, such as surfen, may have therapeutic value.

Xenotropic murine leukemia virus-related virus (XMRV) has been detected in prostate cancer tissues and is therefore a candidate tumor virus. XMRV, which has also been implicated in chronic fatigue syndrome, was first isolated from prostate tissue. Therefore it made perfect sense to determine whether SEVI, which originates from the prostate gland, enhances XMRV infection.

The observation that SEVI enhances XMRV infection is consistent with the possibility that the virus is sexually transmitted. Men with a history of prostatitis or sexually transmitted infections appear to have a higher risk of acquiring prostate cancer. However, the effect of a prostate-derived peptide on XMRV infection might be coincidental: the amyloid fibrils could stimulate infection by other viruses, as noted by the virologists who discovered SEVI:

…the capability to promote the interaction between virions and the cell surface is independent of the viral Env glycoprotein and hence not restricted to retroviruses. Thus, further studies on the role of amyloids in the transmission and pathogenesis of enveloped viruses are highly warranted.

About 30 human diseases, including Alzheimer’s, are associated with deposits of amyloid. Bacterial and fungal infections can also lead to formation of amyloid fibrils – which could explain why sexually transmitted diseases increase the likelihood of acquiring prostate cancer. The ability of these fibrils to enhance infection with different viruses should be examined. It’s possible that different amyloid fibrils are a new general risk factor for certain viral infections.

Hong, S., Klein, E., Das Gupta, J., Hanke, K., Weight, C., Nguyen, C., Gaughan, C., Kim, K., Bannert, N., Kirchhoff, F., Munch, J., & Silverman, R. (2009). Fibrils of Prostatic Acid Phosphatase Fragments Boost Infections with XMRV (Xenotropic Murine Leukemia Virus-Related Virus), a Human Retrovirus Associated with Prostate Cancer Journal of Virology, 83 (14), 6995-7003 DOI: 10.1128/JVI.00268-09

TWiV 67: Wasting deer and the Hulk rabbit

Hosts: Vincent Racaniello, Alan Dove, and Marc Pelletier

Vincent, Alan, and Marc talk about chronic wasting disease of deer caused by prions, blocking the semen-derived enhancer of HIV infection with surfen, and making green transgenic rabbits using a lentiviral vector.

This episode is sponsored by Data Robotics Inc. Use the promotion code VINCENT to receive $50 off a Drobo or $100 off a Drobo S.

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