TWiV 314: Einstein goes viral

On episode #314 of the science show This Week in Virology, Vincent travels to Albert Einstein College of Medicine where he speaks with Kartik, Ganjam, and Margaret about their work on Ebolavirus entry, a tumor suppressor that binds the HIV-1 integrase, and the entry of togaviruses and flaviviruses into cells.

You can find TWiV #314 at

Combination antiviral therapy for hepatitis C

Ledipasvir and SofosbuvirThe Food and Drug Administration has approved the use of a single pill containing two different antiviral drugs for the treatment for hepatitis C. It is the first combination pill approved for the disease, and also the first treatment that does not contain interferon or ribavirin.

The new hepatitis C drug, called Harvoni, is a mixture of the antiviral drugs ledipasvir and sofosbuvir. Ledipasvir (pictured) is an inhibitor of the hepatitis C virus protein NS5A, which has multiple roles in the viral replication cycle that include RNA synthesis and virus particle assembly. The mechanism of NS5A inhibition by ledipasvir is not known. Sofosbuvir is a previously licensed inhibitor that targets the viral RNA-dependent RNA polymerase. It is an analog of the nucleoside uridine, one of the four building blocks of RNA. Sofosbuvir is utilized by the viral RNA polymerase, leading to inhibition of viral RNA synthesis.

The use of single antiviral drugs (monotherapy) to treat RNA virus infections is always problematic because resistance usually arises rapidly. Dual-therapy pills like Harvoni are better, but the best are triple-therapy pills. Triple therapy formulations such as Atripla have been used successfully to treat infections with HIV-1, and presumably there will be mixtures of three antiviral drugs for treating hepatitis C.

Let’s use HIV-1 to illustrate the value of treating infections with multiple antiviral drugs. The HIV-1 viral genome, like that of HCV, is slightly less than 10,000 bases long. Assume that one mutation in the viral genome is needed for drug resistance. If the RNA polymerase mutation rate is 1 out of every 10,000 bases synthesized, then each base in the viral genome is substituted in a collection of 10,000 viruses. An HIV-1 infected person can make as many as 10,000,000,000 virus particles each day, so 1010/104 = one million viruses will be produced each day with resistance to one drug.

If we use two antiviral drugs, developing resistance to both occurs in every 104 x 104 = 108 viruses. In this case 1010/108 = 100 viruses will be produced each day with resistance to two drugs.

If we use three antiviral drugs, developing resistance occurs in every 104 x 104 x 104= 1012 viruses, which is more than what is produced each day.

This is why triple antiviral therapy has been so successful for the treatment of AIDS.

And yes, I’m sure someone has tested Sofosbuvir for inhibition of Ebola virus replication.

TWiV 278: Flushing HIV down the zinc

On episode #278 of the science show This Week in Virology, Vincent, Dickson, Alan, and Kathy discuss disruption of the ccr5 gene in lymphocytes of patients infected with HIV-1.

You can find TWiV #278 at

TWiV 232: Gophers go viral

On episode #232 of the science show This Week in Virology, Vincent meets up with Roberto, Reuben, Lou, and Leslie at the University of Minnesota to talk about their work on HIV-1, APOBEC proteins, measles virus, and teaching virology to undergraduates.

You can find TWiV #232 at

Antimicrobial peptides induced by herpesvirus enhance HIV-1 infection

Langerhans cellsThe risk of being infected with human immunodeficiency virus type 1 (HIV-1) is substantially enhanced in individuals with other sexually transmitted diseases. For example, infection with herpes simplex virus type 2 (HSV-2) increases the risk ratio of acquiring HIV from 2 to 4. Explanations for this increased risk include direct inoculation of HIV-1 into the blood through genital ulcers, and the induction of inflammatory cells by HSV-2 which act as sites of replication for HIV-1. The results of infections carried out in cell culture suggest a biological mechanism for the enhancement of HIV-1 infection by HSV-2.

Langerhans cells (LC) are believed to one of the first cells in which HIV-1 replicates after sexual exposure. LCs are dendritic cells which patrol the mucosal epithelium, taking up and processing antigens and presenting them to T cells in the lymph nodes. These cells express the HIV-1 receptors CD4 and CCR5, but not CXCR4, and can therefore be infected with CCR5-tropic* but not CXCR4-tropic HIV-1. Individuals who do not express CCR5 are resistant to HIV infection. For these and other reasons CCR5-tropic HIV-1 viruses are believed to be ones that transmit infection from one individual to another.

In human skin explant cultures, which contain LCs, co-infection with HSV-2 substantially increased the number of HIV-1 cells. This observation could not be explained by co-infection of individual cells because very few of these were observed in the cultures. When applied to fresh cells, the supernatant of cultures infected with HSV-2 also stimulated the number of HIV-1 infected LCs. These observations suggested that HSV-2 infection stimulates the production of one or more substances from infected cells which in turn improve HIV-1 infection.

Human epithelial and epidermal cells are known to produce antimicrobial peptides such as defensins and cathelicidin. These are short, evolutionarily conserved peptides that inhibit the growth of bacteria, viruses, and fungi. HSV-2 infected keratinocytes were found to produce a number of antimicrobial peptides, but the most important one is called LL-37. This peptide enhanced the expression of HIV-1 receptors CD4 and CCR5 on LCs, leading to increased susceptibility of the cells to HIV-1. Removing LL-37 from the supernatant of HSV-2 infected cells reduces the ability of the medium to stimulate susceptibility to HIV-1.

These findings provide a plausible mechanism by which HIV-1 infection is enhanced by HSV-2. When HSV-2 infects the genital mucosa, the epithelial cells produce LL-37. This antimicrobial peptide enhances the production of CD4 and CCR5 on LCs, allowing more efficient infection by HIV-1. This mechanism is supported by the observation that elevated levels of LL-37 correlate with HIV-1 infection in sex workers.

I wonder why antimicrobial peptides up-regulate CD4 and CCR5. In addition to their antimicrobial properties, the cathelicidins possess chemotactic, immunostimulatory, and immunomodulatory effects, and the upregulation of CD4 and CCR5 are likely part of these activities.

These are exciting findings, and if they are further correlated in humans, they might lead to novel ways of interfering with HIV-1 infection, such as by antagonizing LL-37.

*CCR5 and CXCR4-tropic refer to HIV-1 virions that bind to chemokine receptors CCR5 or CXCR4, respectively, in addition to CD4, to initiate infection.

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.

Click the arrow above to play, or right-click to download TWiV 143 (48 MB .mp3, 66 minutes).

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, by email, or listen on your mobile device with the Microbeworld app.

Links for this episode:

Weekly Science Picks

Rich – Iter – building a fusion reactor
Vincent – American girls sweep at Google science fair (NY Times)

Listener Pick of the Week

JingBees by Rudolph Steiner

Send your virology questions and comments (email or mp3 file) to, or call them in to 908-312-0760. You can also post articles that you would like us to discuss at and tag them with twiv.

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).

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, by email, or listen on your mobile device with the Microbeworld app.

Links for this episode:

Weekly Science Picks

Kathleen – TRIM5 is an innate immune sensor (Nature)
Rich – A Biologist’s Mothers Day Song (YouTube)
Alan – World of Viruses
Vincent – Magnet Balls (Amazon)

Listener Pick of the Week

Chris  – Periodic Tales by Hugh Aldersey-Williams

Send your virology questions and comments (email or mp3 file) to, or call them in to 908-312-0760. You can also post articles that you would like us to discuss at and tag them with 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 in human respiratory tract

An important question about the retrovirus XMRV, which has been implicated in prostate cancer and chronic fatigue syndrome, is where the virus replicates in humans. Such information would provide clues about how infection might be transmitted. To date the virus has been detected in malignant prostate cells and in the peripheral blood mononuclear cells and plasma of patients with CFS. A new study reveals that XMRV is present in respiratory secretions.

Polymerase chain reaction was used to detect XMRV in 267 respiratory samples taken from German patients. One group comprised sputum and nasal swab specimens from 75 travelers from Asia who had respiratory tract infections. The second group consisted of 31 bronchoalveolar lavage samples from patients with chronic obstructive pulmonary disease, while samples from the third group were from 161 immunosuppressed patients with severe respiratory tract infections. The study included 62 healthy controls. It should be noted that none of the patients had been diagnosed with CFS.

XMRV sequences were detected in 3 of 75 samples (2.3%) in group 1, 1 of 31 samples (3.2%) in group 2, and 16/161 (9.9%) in group 3. Six of the XMRV-positive samples in the second group also contained rhinovirus, adenovirus, or pathogenic fungi. The higher rate of detection of XMRV and other microbes in immunosuppressed individuals is not unexpected. The control group contained 2 of 62 samples (3.2%) positive for XMRV.

The presence of XMRV in PBMCs and plasma suggests a blood-borne route of transmission of the virus: transfusions, health care associated needle sticks, and intravenous drug use. Does finding XMRV in the respiratory tract prove that the virus can be transmitted by the respiratory route? No, not until we have other information, including the level of virus in respiratory secretions, and the infectivity of XMRV. In this context it is interesting to note that it was not possible to isolate infectious XMRV from the respiratory tract of the German patients.

Reviewing the transmission of another human retrovirus, HIV-1, is instructive in understanding the pathogenesis of XMRV infection. The main modes of transmission of HIV-1 are sexual, parenteral, and from mother to infant. These routes of transmission are consistent with levels of infectious virus in body fluids (shown in this table). Viral RNA can be detected at several levels of the respiratory tract, but respiratory secretions rarely transmit HIV.

FIsher, N., Schulz, C., Stieler, K., Hohn, O., Lange, C., Drosten, C., & Aepfelbacher, M. (2010). Xenotropic murine leukemia virus-related gammaretrovirus in respiratory tract Emerg. Inf. Dis. : 10.3201/eid1606.100066