A major new feature of the fourth edition of Principles of Virology is the inclusion of 26 video interviews with leading scientists who have made significant contributions to the field of virology. For the chapter on Synthesis of RNA from RNA templates, Vincent spoke with Karla Kirkegaard, PhD, of Stanford University School of Medicine, about her career and her work on picornaviruses.
A small molecule antiviral compound has been shown to protect rhesus monkeys against lethal Ebolavirus disease, even when given up to three days after virus inoculation.
The compound, called GS-5734, is a nucleoside analog. After uptake into cells, GS-5734 is converted to a nucleoside triphosphate (illustrated, bottom panel) which is incorporated by the viral RNA dependent RNA polymerase as it copies the viral genome. However, the nucleoside is chemically different from ATP (illustrated, top) and no further nucleotides can be incorporated into the growing RNA strand. RNA synthesis ceases, blocking production of infectious virus particles.
In cell culture GS-5734 inhibits viral replication at micromolar concentrations, in a variety of human cell types including monocyte-derived macrophages, primary macrophages, endothelial cells, and a liver cell line. The drug inhibits replication of several strains of Zaire ebolavirus, including Kikwit and Makona (from the West African outbreak); Bundibugyo ebolavirus, and Sudan ebolavirus. It also inhibits replication of another filovirus, Marburg virus, as well as viruses of different families, including respiratory syncytial virus, Junin virus, Lassa fever virus, and MERS-coronavirus, but not chikungunya virus, Venezuelan equine encephalitis virus, or HIV-1.
The RNA dependent RNA polymerase of Ebolaviruses has not yet been produced in active form, so the authors determined whether GS-5734 inhibits a related polymerase from respiratory syncytial virus. As predicted, the compound was incorporated into growing RNA chains by the enzyme, and caused premature termination.
Typically tests of antiviral candidates begin in a small animal, and if the results are promising, proceed to nonhuman primates. While a mouse model of Ebolavirus infection is available, the serum from these animals degrades GS-5374. Consequently a rhesus monkey model of infection was used to test the compound.
After intravenous administration of GS-5374, the NTP derived from it was detected in peripheral blood mononuclear cells, testes, epididymis, eyes, and brain within 4 hours. All 12 monkeys inoculated intramuscularly with Zaire ebolavirus died by 9 days post-infection. In contrast, all animals survived after administration of GS-5374 2 or 3 days after virus inoculation. These animals also had reduced virus associated pathology as measured by liver enzymes in the blood and blod clotting. Viral RNA in serum reaches 109 copies per milliliter on days 5 and 7 in untreated animals, and was undetectable in 4 of 6 treated animals.
It is likely that resistant viruses can be obtained by passage in the presence of GS-5734; whether such mutant viruses emerge early in infection, and at high frequency, is an important question that will impact clinical efficacy of the drug. The authors did not detect changes in the viral RNA polymerase gene that might be assoicated with resistance, but further work is needed to address how readily such mutants arise.
These promising results have lead to the initiation of a phase I clinical trial to determine whether GS-5734 is safe to administer to humans, and if the drug reaches sites where Ebolaviruses are known to replicate. However, determining the efficacy of the compound requires treatment of acutely Ebolavirus infected humans, of which there are none. It might be of interest to determine the ability of GS-5734 to clear persistent virus from previously infected individuals.
You can bet that GS-5734 has already been tested for activity against Zika virus.
On 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.
On episode #330 of the science show This Week in Virology, the TWiVers explain how a protein platform assists the hepatitis C virus RNA polymerase to begin the task of making viral genomes.
You can find TWiV #330 at www.microbe.tv/twiv.
On episode #328 of the science show This Week in Virology, the TWiVocateurs discuss how the RNA polymerase of enteroviruses binds a component of the splicing machinery and inhibits mRNA processing.
You can find TWiV #328 at www.microbe.tv/twiv.
The Liberian man who was diagnosed with Ebola virus infection after traveling to Dallas, Texas, was treated with an antiviral drug called brincidofovir. This drug had originally been developed to treat infections with DNA-containing viruses. Why was it used to treat an Ebola virus infection?
Brincidofovir (illustrated) is a modified version of an antiviral drug called cidofovir, which inhibits replication of a variety of DNA viruses including poxviruses and herpesviruses. When cidofovir enters a cell, two phosphates are added to the compound by a cellular enzyme, producing cidofovir diphosphate. Cidofovir is used by viral DNA polymerases because it looks very much like a normal building block of DNA, cytidine. For reasons that are not known, incorporation of phosphorylated cidofovir causes inefficient viral DNA synthesis. As a result, viral replication is inhibited.
Cidofovir was modified by the addition of a lipid chain to produce brincidofovir. This compound (pictured) is more potent, can be given orally, and does not have kidney toxicity, a problem with cidofovir. When brincidofovir enters a cell, the lipid is removed, giving rise to cidofovir. Brincidofovir inhibits poxviruses, herpesviruses, and adenoviruses, and has been tested in phase 2 and 3 clinical trials. The antiviral drug is being stockpiled by the US for use in the event of a bioterrorism attack with smallpox virus.
Ebola virus is an RNA virus, so why was brincidofovir used to treat the Dallas patient? According to the drug’s manufacturer, Chimerix, with the onset of the Ebola virus outbreak in early 2014, the company provided brincidofovir, and other compounds, to the CDC and NIH to determine if they could inhibit virus replication. Apparently brincidofovir was found to be a potent inhibitor of Ebola virus replication in cell culture. Based on this finding, and the fact that the compound had been tested for safety in humans, the US FDA authorized its emergency use in the Dallas patient.
Unfortunately the Dallas patient passed away on 8 October. Even if he had survived, we would not have known if the compound had any effect. Furthermore, the drug is not without side effects and these might not be tolerated in Ebola virus-infected patients. It seems likely that the drug will also be used if other individuals in the US are infected.
Looking at the compound, one could not predict that it would inhibit Ebola virus, which has an RNA genome. RNA polymerases use different substrates than DNA polymerases – NTPs versus dNTPs. NTPs have two hydroxyls on the ribose sugar, while dNTPs have just one (pictured). The ribose is not present in cidofovir, although several hydroxyls are available for chain extension. I suspect that the company was simply taking a chance on whether any of its antiviral compounds in development, which had gone through clinical trials, would be effective. This procedure is standard in emergency situations, and might financially benefit the company.
Update: The NBC news cameraman is being treated with brincidofovir in Nebraska.
On episode #299 of the science show This Week in Virology, Vincent visits the Rocky Mountain Laboratories in Hamilton, Montana and speaks with Marshall Bloom, Sonja Best, and Byron Caughey about their work on tick-born flaviviruses, innate immunity, and prion diseases.
You can find TWiV #299 at www.microbe.tv/twiv.
Vincent, Philip, David, and Priscilla recorded this episode before an audience at the Harvard Virology Program Annual Retreat, where they discussed negative strand RNA viruses, a vaccine against herpes simplex virus type 2, lipidomics of viral infection, and science communication.
The Keynote Speaker at the Harvard Virology retreat is usually an individual, but this year the honor went to TWiV as an example of science communication to the public. Many thanks to members of the Virology Program for a terrific retreat!
Artwork by Silvia Piccinotti, G4
Click the arrow above to play, or right-click to download TWiV 148 (56 MB .mp3, 77 minutes).
Links for this episode:
- Virology at Harvard
- Harvard Virology Retreat 2011 Program (pdf)
- Science in the News
- Lincoln Laboratory, MIT
- Top 10 causes of death (WHO)
- Top 10 infectious disease deaths (Baylor)
- TWiV on Facebook
- Letters read on TWiV 148
- Video of this episode: view below or download (228 MB .mp4)
Weekly Science Picks
Listener Pick of the Week
Jenny – Emerman’s review of Planet of Viruses (PLoS Biology)
Send your virology questions and comments (email or mp3 file) to firstname.lastname@example.org, or call them in to 908-312-0760. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.
Hosts: Vincent Racaniello, Dickson Despommier, and Alan Dove
On episode #62 of the podcast This Week in Virology, Vincent, Dickson, and Alan discuss STEP HIV-1 vaccine failure caused by the adenovirus vector, presence of West Nile virus in kidneys for years after initial infection, adaptation of the influenza viral RNA polymerase for replication in human cells, and the significance of the D225G change in the influenza HA protein.
Click the arrow above to play, or right-click to download TWiV #62 (47 MB .mp3, 66 minutes)
Links for this episode:
- HIV vaccine failure probably caused by adenovirus vector used
- Persistence of West Nile virus in kidneys for years (JID and ProMedMail) (thanks, Lenn!)
- Adaptive strategies of influenza RNA polymerase for replication in humans
- New CDC estimates of 2009 H1N1 infection in US
- Receptor binding specificity of 2009 H1N1 virus
- Distribution of sialic acids in human respiratory tract
Send your virology questions and comments (email or mp3 file) to email@example.com or leave voicemail at Skype: twivpodcast. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.
Hosts: Vincent Racaniello and Dickson Despommier
Vincent and Dick continue Virology 101 with a discussion of how RNA viruses produce mRNA and replicate their genomes.
Click the arrow above to play, or right-click to download TWiV #60 (51 MB .mp3, 71 minutes)
Links for this episode:
- Diagrams of viral RNA synthesis
- Animations of influenza virus and HIV-1 replication
- Video for this episode – see below
Send your virology questions and comments (email or mp3 file) to firstname.lastname@example.org or leave voicemail at Skype: twivpodcast. You can also send articles that you would like us to discuss to delicious and tag them with to:twivpodcast.
Below is a video of TWiV 60, which highlights the diagrams I referred to during the podcast.
Download TWiV 60 video. These videos are slightly larger (800 x 512) than the flash version shown above