On episode #287 of the science show This Week in Virology, Matt Frieman updates the TWiV team on MERS-coronavirus, and joins in a discussion of whether we should further regulate research on potentially pandemic pathogens.

You can find TWiV #287 at www.twiv.tv.

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.

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On episode #286 of the science show This Week in Virology, Vincent and Alan meet up with Julie and Paul at the General Meeting of the American Society for Microbiology in Boston, to talk about their work on the pathogenesis of poliovirus and measles virus.

You can find TWiV #286 at www.twiv.tv.

NP evolutionEvolution proceeds by selection of mutants that arise by error-prone duplication of nucleic acid genomes. It is believed that mutations that are selected in a gene are dependent on those that have preceded them, an effect known as epistasis. Analysis of a sequence of changes in the influenza virus nucleoprotein provides clear evidence that stability explains the epistasis observed during evolution of a protein.

Evolutionary biologist John Maynard Smith used an analogy with a word game to explain how epistasis constrains the evolution of a protein. In this game, single letter changes are made to a four letter word to convert it to another valid word:

WORD->WORE->GORE->GONE->GENE

Although all the intermediates are valid words, the sequence of changes is important. For example, the G in GENE, if introduced into WORD would produce GORD which is not a word. D must be changed to E before W is changed to G. In a similar way mutations in a gene are likely to depend on the changes that have previously taken place.

Whether similar constraints affect protein evolution has been studied with the nucleoprotein (NP) of influenza virus. Between 1968 and 2007, 39 mutations appeared in the NP RNA of influenza virus H3N2. Because sequences of this viral RNA are available each year, it was possible to deduce the order in which these changes appeared in the viral genome (illustrated; figure credit). Plasmids encoding 39 different NP proteins were then constructed which represent viral NP sequences present from 1968 through 2007. All of the NP proteins were found to support similar levels of viral RNA synthesis.

The 39 mutations were then introduced singly into the NP RNA, and RNA synthesis was measured. Three of the altered proteins had large decreases in activity. Their presence also substantially reduced the growth of infectious viruses. However when these NP changes were combined with the amino acid changes that preceded it during evolution, replication was normal. The three NP changes that reduce viral RNA synthesis and replication also decrease the thermal stability of the protein.

These findings show that, from 1968-2007, three amino acid changes were fixed in the influenza virus NP protein whose deleterious effects on protein stability were compensated by previously accumulated changes in the protein. The three amino acids are located in a part of the protein that harbors sequences recognized by T cells. These changes likely allow the virus to escape the host immune response.

Protein stability clearly mediates the epistasis observed in the influenza virus NP protein. It will be important to determine which other protein properties determine the sequence of mutations that are fixed in a viral genome. Influenza viruses are ideal for this work because sequences of all of the viral RNAs are determined for multiple isolates on an annual basis. Studies of what regulates epistasis for other RNA and DNA viruses are also needed to provide an understanding of the constraints of viral evolution.

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Ian Lipkin, Columbia University, New York, and Lyle Petersen, Centers for Disease Control and Prevention, Fort Collins, Colorado, discuss recently emerged pathogens, and how to prepare should their range expand. When asked if MERS-coronavirus would cause the next pandemic, Ian Lipkin responded ‘I don’t have a crystal ball’.

Recorded at the Annual Meeting of the American Society for Microbiology, Boston, MA on 19 May 2014.

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NDV infected glioma cellsThis article was written for extra credit by a student in my recently concluded virology course.

by Nayan Lamba

A recent study by scientists at the Ludwig Center for Cancer Immunotherapy offers a new, multifaceted therapy for destroying tumors. A team of researchers led by Dmitriy Zamarin combined checkpoint blockade, a technique aimed at enhancing antitumor immune responses, with oncolytic viral therapy, a technique that uses viruses to kill tumor cells. By employing the two immunotherapies together, the researchers had more success in destroying tumor cells than they have had while investigating each therapy independently.

While checkpoint blockade has been effective as a therapy against some tumors, its major drawback seems to be an inability to destroy strongly immunosuppressing tumors that evade immune system detection. While many oncolytic viruses have also had success as antitumor agents, their impact thus far has been limited to locally restricted tumors. Scientists major concern about oncolytic viruses has thus been whether they must be injected at all possible tumor sites in order to combat metastatic tumors. In an innovative approach aimed at overcoming the weaknesses of the two independent therapies, Zamarin and his team were able to destroy previously resistant tumor cells.

The researchers initially injected mice with melanoma tumor cells, followed by injection of Newcastle disease virus (NDV) directly into these tumor sites. Newcastle disease virus is an avian virus that is non-pathogenic in humans and capable of inducing a robust immune response. Initial injection of NDV resulted in a pronounced inflammatory response in the mice, with increased activation of both innate and adaptive players of immunity. What is perhaps most striking about the data is that despite direct injection of NDV at a local tumor site, increased inflammation was also observed in sites distal and contralateral to tumor injection. The researchers noted tumor growth delay at sites both local and distant to sites of injection, indicating a potential for use of NDV in targeting metastatic tumors. While this observation was certainly promising, the researchers noted that complete, long-term destruction of distant tumor cells was seen in only 10% of animals. The researchers attributed this low level to the increased immunosuppression performed by the distant tumors. This suggestion was based on their observation that the distant tumor sites exhibited increased activity of CTLA-4 cells, which down-regulate the immune system.

While a more traditional, unilateral approach employing oncolytic viruses would have stopped here, the researchers instead proceeded to couple NDV injection with antibodies to CTLA-4 cells. They hypothesized that because NDV caused an increased level of inflammation at distant tumor sites, the anti-CTLA-4 antibodies might have more of an effect if they had been administered without NDV. Indeed, the researchers found that NDV coupled with anti-CTLA-4 resulted in long-term tumor suppression at sites both local and distant to NDV injection. By increasing the inflammatory response via NDV injection, they made the immune cells more receptive to the anti-CTLA-4 antibodies. Through a combination of oncolytic virus therapy and checkpoint blockade, the researchers overcame the limitations faced when each one is employed independently.

What is perhaps most promising about this therapy is that it has also proved effective against tumors that have previously shown resistance to oncolytic viral therapies. For example, TRAMP C2 prostrate adenocarcinoma cells previously showed resistance to lysis by NDV in vitro, unlike the melanoma cells discussed above. Yet, despite this resistance, when both therapies were employed on the adenocarcinoma cells in vivo, researchers noted distant tumor regression and long-term survival, just as they did with the melanoma cells. When they examined these adenocarcinoma cells in vitro, they noted an increased inflammatory response. They noted an up-regulation of MHC I molecules in all cells, even cells that were not infected with NDV. MHC I cells are important players of the immune system, responsible for presenting fragments of virus at the surface of infected cells so that the body can recognize when a cell is infected and subsequently destroy that cell. By demonstrating that a tumor cell does not need to be permissive to a virus in order to be a target for therapy, Zamarin’s approach greatly expands the potential applicability of NDVs and other oncolytic viruses. It seems that what is most important to tumor suppression is the virus-generated inflammatory response and the increased tumor immogenicity that this approach facilitates.

The clinical potential of this double-therapy in humans is particularly exciting to me, especially in light of the fact that both oncolytic therapies and checkpoint blockade have independently been successful in combating tumors in humans. This observation certainly suggests that the two therapies may prove even more effective when combined in humans. Indeed, such a clinical trial is already underway in humans; a current study is studying the effects of ipilimumab, a CTLA-4 target, administered with a herpes simplex oncolytic virus, in the treatment of melanoma. It will be interesting to see how the double-therapy plays out in a human population, and how these results affect the future use of oncolytic viruses.

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On episode #285 of the science show This Week in Virology, Vincent meets up with XJ Meng and Sarah McDonald at Virginia Tech to talk about their work on viruses of swine and rotaviruses.

You can find TWiV #285 at www.twiv.tv.

variola virusLater this month (May 2014) the World Health Assembly will decide whether to destroy the remaining stocks of variola virus – the agent of smallpox – or to allow continued research on the virus at WHO-approved laboratories.

After the eradication of smallpox in 1980, the World Health Organization called for destruction of known remaining stocks of variola virus. The known remaining stocks of the virus are closely guarded in the United States and Russia. These consist not of a single vial of the virus, but of hundreds of different strains, many of which have not been fully characterized, nor has their genome sequence been determined.

It can be argued that there still remains a good deal of work to be done on variola virus, including development of newer diagnostic tests, and identification of additional countermeasures (antivirals and vaccines have been stockpiled in the US). Damon, Damaso, and McFadden have written a summary of the research on variola virus that should be done. We also discussed whether the remaining variola virus stocks should be destroyed on episode #284 of This Week in Virology.

We are interested in what readers of this blog think about this issue – please fill out the poll below.

Destruction of variola virus
Should all known remaining stocks of variola virus, agent of smallpox, be destroyed?

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On episode #284 of the science show This Week in Virology, the TWiV team discusses how skin scarification promotes a nonspecific immune response, and whether remaining stocks of smallpox virus should be destroyed.

You can find TWiV #284 at www.twiv.tv.

cultured cellsThis week’s question comes from a graduate student studying virology, who writes:

My professor recently said that really, the MOI doesn’t matter in a culture, it is the concentration of viral particles in the media that matters. Ie: if you have 10 million cells or one cell, but you are infecting the plate with 5mL of 100 million viral particles/mL, then the amount of virus interacting with each cell is not different in either scenario (pretending that it isn’t nearly impossible for that single cell to survive in culture alone). I argued with him, saying that the cytotoxicity to the single cell would certainly be increased. He then said that a student hadn’t argued with him about that in his 15 years of teaching and I promptly decided to get some evidence before I continued the discussion.

I’m not actually sure which side is correct. I know that concentration is certainly a large determinant for infectious events/cell. But, it is hard for me to understand why MOI wouldn’t be more important? The more I think about it the more I think that I may be wrong. But if you have two plates with equal numbers of cells, and you add 5 mL of media to one and 50mL of media to the other – assuming that the media is 100 million infectious particles/mL – would the higher MOI plate not result in more infectious events per cell?

My reply: What first jumps out at me is the fact that the professor is using the no one ever argued with me about that excuse to say that he/she is right. That is the exact role of a student, to ask questions, and it should never be discouraged. Students can ask the best questions because they are frequently unencumbered by the bias of a field.

Please tell your professor that both multiplicity of infection and concentration of viral particles matter, for different reasons. The multiplicity of infection (MOI) is the number of virus particles added per cell. If you add one million virus particles to one million cells in a culture plate, the MOI = 1. If you add ten million virus particles to one million cells, the MOI is 10.

However, if one million virus particles are added to one million cells, each cell will not be infected with one virus particle. How many cells are uninfected, or receive 1, 2, or more virus particles is determined by the Poisson distribution. At an MOI of 1, 37% of the cells are uninfected, 37% receive 1 particle, 18% receive 2 particles, and so on.

In theory, the number of particles that infect each cell is controlled by the MOI, not the virus concentration. However, when the concentration of virus particles is very low, attachment to cells will take a very long time. This is because virus attachment is governed by the concentrations of free virions and host cells. The rate of attachment can be described by the equation

dA/dt = k[V][H]

where [V] and [H] are the concentrations of virions and host cells, respectively, and k is a rate constant.

For a 6 cm culture dish with an area of 113 square cm, we typically infect with virus in a volume no greater than 0.1 – 0.2 ml. In this way virus attachment to cells will be essentially complete within 1 hr at 37 degrees C. If the same amount of virus were added in 10 ml of medium, the attachment would take much longer; however because the MOI is the same in both cultures, at the end of the adsorption period the number of infected and uninfected cells in both cultures would be the same.

To answer the reader’s last question:

But if you have two plates with equal numbers of cells, and you add 5mL of media to one and 50mL of media to the other – assuming that the media is 100 mill infectious particles/mL – would the higher MOI plate not result in more infectious events per cell?

The answer is yes – assuming you wait long enough for the viruses in the more dilute culture to attach to cells.

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