TWiV 324: Viruses in the miR may appear more numerous

On episode #324 of the science show This Week in Virology, Lee joins the TWiV team to discuss the value of post-doctoral training, and how a cellular microRNA assists in the replication of hepatitis C virus.

You can find TWiV #324 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.

What price antiviral drugs?

SofosbuvirThe Federal Drug Administration of the US approves new drugs solely on the basis of safety and effectiveness, with no value assessment. Pharmaceutical companies may set their drug prices based mainly on what the market will bear. Nevertheless, the announcement that Gilead Sciences would price their just-approved, anti-hepatitis C virus (HCV) drug sofosbuvir (Solvaldi) at $84,000 for 12 weeks of treatment was met with considerable complaints.

Solvaldi is a member of a class of antiviral drugs called nucleoside analogs. They act as chain terminators and inhibit viral RNA synthesis. When the viral RNA polymerase is copying the viral RNA, to enable the production of more virus particles, it normally uses the pool of ATP, UTP, GTP, and CTP to produce more RNA. When Solvaldi is incorporated into the growing RNA chain by the viral enzyme, no additional triphosphates can be added, because the drug contains a fluorine atom at the 2′-position of the ribose. Its presence inhibits addition of the next nucleoside by the polymerase to the 3′-OH. Viral RNA synthesis therefore stops, and production of virus particles is inhibited. For more information on chain terminators, see my virology lecture on antivirals.

Gilead believes that the price of the drug is fully justified: a spokesperson said “We’re just looking at what we think was a fair price for the value that we’re bringing into the health care system and to the patients.”

It could cost up to $300,000 to treat patients with chronic HCV infection using less effective and more difficult to tolerate regimens. The potential benefit of a cure for patients with liver disease is clear, as the virus is the main reason that nearly 17,000 Americans are waiting for a liver transplant. The need for a well-tolerated, effective regimen is equally critical for people infected with HIV and HCV, because having both infections accelerates liver damage.

Despite these arguments, the high price will be a significant barrier for many, especially those in limited and fixed-budget programs such as Medicare and Medicaid. A panel of experts in San Francisco estimated that switching HCV infected Californians to Sovaldi would raise annual drug expenditures in the state by at least $18 billion.

Gilead has agreed to help U.S. patients pay for Sovaldi if they cannot afford it, or help patients obtain drug coverage. The company also plans to charge substantially less for a course of treatment in India ($2000 for the 12 week course), Pakistan, Egypt ($990 for the 12 week course), and China, where most people infected with HCV live. These deals have prompted some to ask if the US is being forced to subsidize the cost of the drug worldwide. I personally do not object to helping other countries solve their HCV problem.

What is a fair price for a drug that can eliminate HCV infection? Gilead paid more than $11 billion in 2011 to acquire the company that developed Sovaldi, and it is reasonable for them to recoup that investment. Andrew Hill of the Department of Pharmacology and Therapeutics at Liverpool University estimates the manufacturing cost of a 12 week course of treatment with this drug to be $150 to $250 per person. The answer to our fair price question must lie somewhere between these extremes.

There are parallels between Sovaldi (and other new anti-HCV drugs in the pipeline) and the initially expensive antivirals that were introduced ~20 years ago to treat HIV. Anti-retrovirals revolutionized the treatment of a chronic, lethal infection that is major global health problem, and the anti-HCV drugs could have the same effect. But there are also important differences: based on the number of infected individuals, HCV is a much larger public health threat than HIV. Furthermore, the new HCV antivirals can eliminate the virus completely, whereas anti-HIV drugs only suppress virus replication, so they must be taken (and paid for) for life.

At some point in the future competition among pharmaceutical companies and manufacturers of generic drugs should make it possible to treat everyone infected with HCV with affordable, curative antivirals. If the cost and efficiency of diagnosis and drug delivery keeps pace, it might be possible to eradicate HCV. That accomplishment might well be priceless.

TWiV 274: Data dump

On episode #274 of the science show This Week in Virology, the TWiV team discusses recent cases of polio-like paralysis in California, and the virome of 14th century paleofeces.

You can find TWiV #274 at

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 235: Live in Edmonton, eh?

Episode #235 of the science show This Week in Virology was recorded before an audience at the 2nd Li Ka Shing Institute of Virology Symposium at the University of Alberta, where they spoke with Dave, Stan, and Lorne about their work on poxvirus vaccines and recombination, an enveloped picornavirus, antivirals against hepatitis B and C viruses, and supporting virology research in Alberta.

You can find TWiV #235 at

Did hepatitis C virus originate in horses?

Dog and horseAbout 2% of the world’s population is chronically infected with hepatitis C virus (HCV). This enveloped, positive-strand RNA virus was discovered in 1989, but serological and phylogenetic evidence indicates that it has been infecting humans for hundreds of years, perhaps as long ago as the 14th century. All human viral infections most likely originated in non-human species, but the progenitor of HCV is not known. Recent evidence suggests that horses might have been the source of HCV in humans.

For many years there were no known non-human relatives of HCV until canine hepacivirus was discovered in dogs (we discussed this virus on TWiV #137). However two subsequent studies failed to reveal additional evidence for CHV infection of dogs. In one study, no antibodies to CHV were found in sera from 80 dogs in New York State, and in a second study, PCR failed to detect CHV nucleic acid sequences in 190 samples from dogs in Scotland. Samples from rabbits, deer, cows, cats, mice, and pigs were also negative for CHV. However both groups found evidence for infection of horses. These viruses have been called non-primate hepaciviruses (NPHV).

In one study carried out on horses in New York State, 8 of 103 samples were found to contain antibodies to NPHV. Complete viral genomes were identified from all 8 horses. Most are genetically distinct from CHV, but one viral sequence, obtained from a pool of sera from New Zealand horses, is nearly identical to CHV. NPHV was also detected by PCR in sera from 3 of 175 Scottish horses. Separate serum samples obtained from one horse 5 months apart were positive for viral RNA, indicating persistent infection. None of the horses had any evidence of clinical hepatitis or any other illness.

These results from geographically distinct areas suggest that horses are a reservoir of NPHV. It seems likely that dogs might acquire NPHV infection from horses, as there are opportunities for contact between the two animals on farms or in kennels. Additional NPHV isolates from horses must be studied to confirm this hypothesis.

It will be important to determine if horse NPHV was the source of human HCV. This is theoretically possible because horse products, such as serum containing antibodies to pathogens or toxins, have been injected into humans. There are six genotypes of HCV, each of which is believed to have emerged at different times and geographic locations. Whether their emergence represent different cross-species transmissions, as is the case with the different groups of HIV-1, remains to be determined.

I also wonder how horses originally acquired NPHV. Perhaps it was transmitted to them from another species via a vector bite, such as a mosquito – but from what species?

Great ape protection act

chimpanzee HamI received the following email today from Judith S. Bond, President of the Federation of American Societies for Experimental Biology (FASEB):

Dear Colleague,

We need your help to counter a serious threat to the humane use of animals in research. The Great Ape Protection and Cost Savings Act (S 810), which would prohibit the use of chimpanzees in medical research, may be voted on in the Senate this week (it was approved by a Senate committee in July)! Passage of this bill could have devastating consequences for ongoing research into human diseases such as hepatitis C, as well as studies benefiting the great apes themselves. Even if you do not work with great apes, you should be concerned about this bill because it would end research deemed by the Institute of Medicine (IOM) to be ethically sound and scientifically important and could pave the way for legislation to ban research with other species

Those who oppose the use of animals in research are making an aggressive effort to get this bill passed before Congress goes home for the year. We must let them know that chimpanzees are important animal models for research. Please take action now by going to to send an email to your Senators urging them to oppose the Great Ape Protection and Cost Savings Act.

You can find the text of this bill on this webpage (the pdf link at the top of the page provides the most readable version of the bill). Great apes include the chimpanzee, bonobo, gorilla, orangutan, and gibbon. Invasive research is any research that may cause death, injury, pain, distress, fear, or trauma.

According to the text of the bill, the chimpanzee is the only great ape used for invasive research in the United States, where there are approximately 1000 housed in laboratories.

The bill cites the Institute of Medicine and National Research Council report entitled “Chimpanzees in Biomedical and Behavioral Research: Assessing the Necessity” which concluded that most current use of chimpanzees for biomedical research is unnecessary.  It states that research on hepatitis C antiviral drugs, respiratory syncytial virus, future monoclonal antibody therapies, or a therapeutic hepatitis C virus vaccine, does not require chimpanzees, and that ‘the use of a combination of non-chimpanzee methods for the development of monoclonal antibody therapies may make research on the chimpanzee largely unnecessary; and non-chimpanzee models, if further improved, may reduce or obviate the need for the continued use of the chimpanzee for prophylactic hepatitis C vaccine research.’

Presumably the authors of the bill refer in part to work directed on developing a mouse model for HCV infection. However, as indicated by the bill’s language, it is not yet clear if these models will supplant the chimpanzee for HCV research.

The purpose of this act is to phase out invasive research on great apes and the use of Federal funding of that research, both in and outside of the United States. All existing chimpanzee protocols must be terminated within three years of passing of the bill, and once the bill has been passed, no new chimpanzee experiments may be started.

There is an escape clause – if, after three years have passed, it is determined that a new disease requires research on chimpanzees, the Great Ape Task Force will be created to evaluate that need.

If this act had been passed in the 1950s, it might not have been possible to develop poliovirus vaccines. While transgenic mice recapitulate much of poliovirus pathogenesis, they are not orally susceptible to infection (unless the interferon system is disabled) and therefore cannot reliably be used to test protection conferred by immunization.

It seems premature to pass an act banning research on chimpanzees. These animals are needed for testing anti-HCV therapies and vaccines, and as stated in the bill, it is not yet clear if other animal models will replace chimpanzees. It seems prudent to wait until we have a suitable animal model for HCV (and other infectious and non-infectious diseases which currently require chimpanzees) before outlawing the use of this animal in research.

TWiV 188: Haggis, single malt, and viruses

On episode #188 of the science show This Week in Virology, Vincent travels to Scotland to meet with members of the Centre for Virus Research at the University of Glasgow to discuss their work on hepatitis C virus and jaagsiekte sheep retrovirus.

You can find TWiV #188 at

TWiV 180: Throwing IFIT at flu and holding a miR to HCV

On episode #180 of the science show This Week in Virology, Vincent, Alan, and Rich review association of an interferon-induced protein with severe influenza, and stabilization of HCV RNA by a microRNA.

You can find TWiV #180 at