Interview with Thomas London

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. These in-depth interviews provide the background and thinking that went into the discoveries or observations connected to the concepts being taught in this text. Students will discover the personal stories and twists of fate that led the scientists to work with viruses and make their seminal discoveries.

For the chapter on Infections of Populations, Vincent spoke with Thomas London, MD, of the Fox Chase Cancer Center, about his career and his work on hepatitis B virus.

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.

Hepatitis B viruses in bats

hepadnaviridae virionHepatitis B virus (HBV, illustrated) is a substantial human pathogen. WHO estimates that there are now 240,000,000 individuals chronically infected with HBV worldwide, of which 25% will die from chronic liver disease or hepatocellular carcinoma. The hepatitis B virus vaccine is highly effective at preventing infection. Because there are no known animal reservoirs of the virus, it is believed that HBV could be globally eradicated. The recent finding of HBV in bats raises the possibility of zoonotic introduction of the virus.

Serum and liver samples from 3,080 bats from Panama, Brazil, Gabon, Ghana, Germany, Papua New Guinea, and Australia were screened for HBV-like sequences by polymerase chain reaction (PCR). Ten positive specimens were found from three bat species: Uroderma bilobatum from Panama, and Hipposideros cf. ruber and Rhinolophus alcyone from Gabon. The complete viral genome sequence was determined for 9 of the positive specimens. Phylogenetic analysis revealed that the bat viruses form three different lineages, and that each virus differs by at least 35% from known hepadnaviruses.

The virus from Hipposideros cf. ruber has been named roundleaf bat HBV, while those from Rhinolophus and Uroderma have been named horshoe bat HBV, and tent-making bat HBV.

Viral DNA in the liver of Hipposideros bats was found to be higher than in other organs or serum. Some lymphocyte infiltration was observed in the liver of these animals, as well as deposits of viral DNA within hepatocytes. These observations indicate that the bat HBV viruses likely replicate in the bat liver and cause hepatitis.

Serological studies revealed that hepadnaviruses are widespread in Old World bats: antibodies against bat hepadnaviruses were detected in 18% of hipposiderid bats and 6.3% of rhinolophid bats.

An important question is whether these three bat hepadnaviruses can infect human cells. Only tent-making bat HBV could infect primary human hepatocytes, which occurred via the human HBV cell receptor, sodium taurocholate cotransporting polypeptide. However serum from humans that had been immunized with HBV vaccine did not block infection of human hepatocytes with this virus.

These observations show that viruses related to human HBV are replicating in the liver of bats. Earlier this year another hepadnavirus was identified in long-fingered bats (Miniopterus fuliginosus) in Myanmar. The complete genome sequence was obtained and virus particles were observed in bat liver tissues.

The finding of hepadnaviruses in bats raise many interesting questions. The first is whether human HBV originated by infection with bat HBV, either by consumption of bat meat or another mode of transmission. How long ago this occurred is not known. It has been suggested that HBV has been in humans for at least 15,000 years. Some avian species contain avihepadnaviral sequences integrated into their genome, indicating that these viruses originated at least 19 million years ago.

These findings also raise many questions about the pathogenesis of hepadnaviral infection in bats, including the mode of transmission (in humans, the virus is transmitted by exposure to blood, e.g. by injection or during childbirth), and whether chronic infections can occur as they do in humans.

Finally it is interesting to consider the zoonotic potential of tent-making bat HBV, which can infect human cells. Because bat hepadnaviruses are genetically distinct from HBV, current serological and nucleic acid screening programs would not detect human infections. The authors suggest that human and non-human primate sera from areas in which these bat viruses were isolated should be screened using assays that detect the bat hepadnaviruses. Without such information we do not know if these viruses currently infect humans.

Treating hepatitis C by blocking a cellular microRNA

HCV UTRMiravirsen is a drug that binds to and blocks the function of a cellular microRNA called miR-122 that is required for the replication of hepatitis C virus (HCV). Treatment of chimpanzees chronically infected with HCV with this drug leads to suppression of viral replication. The results of a phase 2b human clinical trial in HCV infected humans indicate that Miravirsen reduces levels of viral RNA without evidence for viral resistance. I asked virologist Stan Lemon (who appeared recently on TWiV 235) his opinion of these findings.

Are you surprised that the antiviral effect of Miravirsen is long lasting?

The Janssen study published in NEJM basically recapitulated what Lanford had observed in HCV-infected chimps treated with the compound, with a very slow onset of antiviral effect, and then a very slow rebound as well. This probably reflects the pharmacokinetics and very high stability of the locked nucleic acid compound, and the time required to sequester endogenous miR-122 – changes in serum cholesterol also move very slowly. I think this is why the antiviral effect (and cholesterol effect) are long-lasting.

Is it surprising that no resistance to Miravirsen was observed?

As for the lack of resistance, it doesn’t surprise me much. This was observed in the chimps as well. The virus is really dependent upon miR-122 for its replication, and can’t readily mutate around it – the requirement for miR-122 reflects more than just the stabilizing effect of miR-122 on the viral genome, as we showed in a recent PNAS paper (Li et al., Proc. Nat’l. Acad. Sci U.S.A., 110:1881-6, 2013) written in follow-up to our earlier demonstration of the stabilizing effect of the miRNA on the HCV genome (Shimakami et al., Proc. Nat’l. Acad. Sci U.S.A. 109: 941-6, 2012, that you reviewed in TWIV 180) – what we know and don’t know about the mechanism of action is summarized in an “opinion” piece now in press in RNA Biology.

Do you think this drug will ultimately get FDA approval?

Given issues of resistance, relapse, and poor pan-genotype coverage with direct-acting antivirals for HCV, all of this should bode well for Miravirsen. However, it has issues like almost all the new therapies under evaluation.

First, the spaghetti plots in the Janssen paper show large variation in the response of individual patients, with some having little effect when receiving Miravirsen. This is unlike studies with enzyme inhibitor antivirals, and I am not aware of any good reason for it other than potential variation in endogenous miR-122 abundance.

A second and greater issue is the cancer concern. Most hepatocellular carcinomas (except those associated with HCV, interestingly enough) demonstrate significant reductions in miR-122 abundance, and miR-122 can reverse some malignancy-associated features of transformed hepatocytes in vitro – thus, miR-122 seems to act much like a tumor suppressor in the liver.  miR-122 knockout mice develop normally but have a high incidence of hepatocellular carcinoma. I think this poses real problems for the development of Miravirsen. While one could reasonably argue that short-term exposure to the antagomir is very different than gene knockout, the patients being treated are those at the highest risk for HCC – particularly if there is advanced fibrosis or cirrhosis, which characterizes those most in need of treatment. It is also clear that HCC can manifest itself in patients AFTER therapeutic elimination of the virus. The risk is most certainly greatly reduced, but it is not zero (HCC develops very slowly, and in a multi-centric fashion), and with the evidence that the drug has relatively long-lasting effects on cholesterol (as well as the virus), I think the developers of Miravirsen may find it difficult to defend against future claims that the drug contributed to the development of HCC in some cases. There isn’t a good way to de-risk this, to show that this theoretical concern is not real, and this must be worrying the regulatory authorities – especially since there are now many alternative therapies under evaluation that don’t carry this risk, some of which are looking very good in combination with each other (e.g., advanced NS3 inhibitors, NS5A inhibitors, and nucs).

TWiV 85: Hepatitis C virus with Professor Michael Gale

Hosts: Vincent Racaniello and Michael Gale

On episode 85 of the podcast This Week in Virology, Vincent and Michael Gale discuss the origin, pathogenesis, prevention, of hepatitis C virus, and how it evades innate immune responses.

This episode is sponsored by Data Robotics Inc. Use the promotion code TWIVPOD to receive $75-$500 off a Drobo.

Click the arrow above to play, or right-click to download TWiV #85 (40 MB .mp3, 56 minutes)

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

Links for this episode:

  • The Gale Laboratory at the University of Washington
  • Incredible view from the Gale laboratory (jpg)
  • Evasion and disruption of innate immune signalling by hepatitis C and West Nile viruses (review)
  • New potent HCV inhibitor
  • HCV virion and genome structures at ViralZone

Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv 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.

TWiV 80: How much X could a woodchuck chuck?

Hosts: Vincent Racaniello, Alan Dove, Rich Condit, and Michael Bouchard

Vincent, Alan, and Rich speak with Michael Bouchard about hepatitis B virus discovery, replication, and pathogenesis.

This episode is sponsored by Data Robotics Inc. Use the promotion code TWIVPOD to receive $75-$500 off a Drobo.

Win a free Drobo S! Contest rules here.

Click the arrow above to play, or right-click to download TWiV #80 (58 MB .mp3, 80 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email.

Links for this episode:

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Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv 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.