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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On episode #283 of the science show This Week in Virology, Jens Kuhn speaks with the TWiV team about filoviruses, including the recent Ebola virus outbreak in Guinea.

You can find TWiV #283 at www.twiv.tv.

1918 influenza mortalityThe 1918 influenza pandemic was particularly lethal, not only for the very young and the very old (as observed for typical influenza), but unexpectedly also for young adults, 20 to 40 years of age (pictured). It has been suggested that the increased lethality in young adults occurred because they lacked protective immunity that would be conferred by previous infection with a related virus. Reconstruction of the origins of the 1918 influenza virus provides support for this hypothesis.

Analysis of influenza virus genome sequences using a host-specific molecular clock together with seroarchaeology (analysis of stored sera for the presence of antibodies to influenza virus) indicates that the 1918 H1N1 virus arose ~1915 by reassortment of an avian influenza virus with an H1 virus that had previously emerged around 1907. The 1918 virus acquired the HA gene from the 1907 virus, and the NA gene and internal protein genes from an avian virus. This 1918 virus also infected pigs, in which descendants continue to circulate; however the human 1918 virus was displaced in 1922 by a reassortant with a distinct HA gene.

Seroarchaeology and mortality data indicate that an influenza pandemic in 1889-1893 was caused by an influenza H3N8 virus. This virus appears to have circulated until 1900, when it was replaced by a H1N8 virus (the N8 gene originating from the previously circulating H3N8 virus).

How do these events explain the unusual mortality pattern of the 1918 influenza A virus? High mortality among 20-40 year old adults might have been a consequence of their exposure to the H3N8 virus that circulated from 1889-1900. This infection provided no protection against the 1918 H1N1 virus. Protection of other age groups from lethal infection was likely a consequence of childhood exposure to N1 or H1 containing viruses (this may also have resulted in the lower than usual mortality in the elderly population). Influenza is typically highly lethal in very young children due to lack of immunologic memory.

These observations suggest that childhood exposure to influenza virus is a key predictor of virulence of a pandemic strain. Antibodies against the stalk of the HA protein protect against severe disease, but only within groups of HA subtypes (HA groups are determined by phylogenetic analysis). In 1918, antibodies against a group 2 HA subtype virus (H3) did not protect against severe disease caused by a group 1 HA subtype virus (H1). Childhood exposure might also determine mortality of seasonal influenza. For example, the high virulence of currently circulating H3N2 influenza viruses in those older than 65 years might be a consequence of infection with an H1N1 virus at a young age.

This logic can also explain mortality caused by influenza H5N1 and H7N9 viruses. Most fatalities caused by H5N1 viruses (the H5 is a group 1 HA) have been in individuals who were infected as children with an H3 virus (group 2 HA). Most fatalities caused by H7N9 viruses (group 2 HA) have occurred in individuals who were infected as children with H1N1 or H2N2 viruses (group 1 HA).

The practical consequence of this work are clearly stated by the authors:

Immunization strategies that mimic the apparently powerful lifetime protection afforded by initial childhood exposure might dramatically reduce mortality due to both seasonal and novel IAV strains.

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On episode #282 of the science show This Week in Virology, the TWiV team reviews a meta-analysis of clinical trial reports on using Tamiflu for influenza, and suggestions on how to rescue US biomedical research from its systemic flaws.

You can find TWiV #282 at www.twiv.tv.

influenza-reassortmentThis week’s virology question comes from Eric, who writes:

I’m working on an MPH and in one of my classes we are currently studying the influenza virus. I’d forgotten that the genome is in 8 separate parts. Curious, I’ve been searching but can’t find any information as to why that is?

What evolutionary advantage is conferred by having a segmented genome?

Terrific question! Here is my reply:

It’s always hard to have answers to ‘why’ questions such as yours. We answer these questions from a human-centric view of what viruses ‘need’. We might not be right. But I’d guess there are at least two important advantages of having a segmented RNA genome.

Mutation is an important source of RNA virus diversity that is made possible by the error-prone nature of RNA synthesis. Viruses with segmented genome have another mechanism for generating diversity: reassortment (illustrated).

An example of the evolutionary importance of reassortment is the exchange of RNA segments between mammalian and avian influenza viruses that give rise to pandemic influenza. The 2009 H1N1 pandemic strain is a reassortant of avian, human, and swine influenza viruses.

Having a segmented genome is another way to get around the limitation that eukaryotic mRNAs can only encode one protein. Viruses with segmented RNA genomes can produce at least one protein per segment, sometimes more. There are other ways to overcome this limitation – for example by encoding a polyprotein (picornaviruses), or producing subgenomic RNAs (paramyxoviruses).

Other segmented viral genomes include those of reoviruses, arenaviruses, and bunyaviruses.

There are various ways to achieve genetic variation and gene expression, and viruses explore all aspects of this space.

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On episode #281 of the science show This Week in Virology, Vincent meets up with Peter L. Salk to talk about development of the first poliovaccine, eradication of poliomyelitis, and Jonas Salk’s 100th birth anniversary.

You can find TWiV #281 at www.twiv.tv.

TWiV 280: Post viral

13 April 2014

On episode #280 of the science show This Week in Virology, the TWiVmeisters answer listener email about the NEIDL, negative results, patenting MERS-coronavirus, human papillomavirus transmission, canine distemper virus, and much, much more.

You can find TWiV #280 at www.twiv.tv.

Unraveling the NEIDL

11 April 2014

Threading the NEIDLThe NEIDL (National Emerging Infectious Diseases Laboratory) at Boston University is a newly constructed biosafety level 4 facility which can be used to study the most dangerous human pathogens. The facility is amazingly safe, as we documented in our film about the facility, Threading the NEIDL. Some members of the Boston City Council think otherwise and have moved to stop the facility from opening.

In January 2014 a draft ordinance was introduced to the Boston City Council that would prohibit BSL-4 research within the city limits. The first public hearing on the proposed ordinance is scheduled for Wednesday, April 16, 2014 at Boston City Hall. Members of the public are invited to attend and testify. I encourage you to read the proposed ordinance which is available online. If you live in the Boston area and have a view on this ordinance, you might consider attending the public hearing. The Boston City Council Calendar of Meetings should be consulted for scheduling changes.

The American Society for Microbiology has provided this statement on the ordinance:

As background information you should be aware that In 2007, the ASM filed an Amicus brief with the Supreme Court of Massachusetts on a case involving a BSL-4 laboratory affirming the importance and safety of BSL-4 research laboratories. That same year, the Society also provided similar testimony before the U.S. House of Representatives.

We believe that scientists should be aware of events that can impact science in their region and have the opportunity to voice their opinions on actions that could affect them, directly or indirectly. We urge any members who are inclined to attend.

The Boston City Council Ordinance seems ill-advised, especially since the NEIDL has gone through a supplemental risk assessment process, and has been blessed by two independent scientific groups (the National Research Council and the NIH Blue Ribbon Panel), and prevailed in Federal Court.

While it might seem frightening to have a BSL-4 facility within a major city, after having toured the NEIDL I can say with confidence that the precautions taken to prevent release of pathogens, both physical and operational, are second to none. As Elke Muhlberger noted during our tour, it is the safest place to work on Earth. I strongly recommend that the Boston City Council members view Threading the NEIDL to learn why it does not make sense to prevent opening of this facility.

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HIV binding CD4 and ccrOn the science show This Week in Virology we receive many questions and comments, which are read every week. I also get many questions here on virology blog, which I tend to answer by email. However I think that everyone could benefit from these questions, so I’ve decided to post one here each week along with my answer.

This week’s question is from Joseph, who wrote:

I’m relatively new to virology or anything biology-related. Hell, I’m studying computer science as an undergrad at the moment; however, there’s something about virology that fascinates me – the simplistic fact that we can’t cure viruses, which are less complex than bacterium (in which we can treat, and they’ll eventually pack their bags and leave).

I’ll get to my question … since most, if not all, cells in the body replicate and reproduce and none of them merge, why do our cells let virions in? You would think after years of viral/immune system encounters, our bodies would have adapted to repelling these viruses off. I understand it’s probably much more complicated than that, but I would love to hear your answer. Does it have anything to do with virions’ size being so small?

This is a great question. In fact, I had a similar question on a midterm examination in my virology course. I phrased it this way: Could cells evolve to not have receptors for binding viruses?

I sent this answer to Joseph:

Viruses get into cells by binding to proteins on the cell surface – viruses have evolved to do this: they are safecrackers.

You would think that the cells would evolve to change these proteins – and you would be right. Over thousands of years, the cell proteins change, so the viruses can’t bind anymore.

But guess what? The viruses change right back so that they can bind to the cell protein once more.

Now you might ask: why doesn’t the cell get rid of that surface protein? The answer there is that they are needed for the cell, so they can’t be removed.

There seems to be one exception to the last statement: about 4-16% of people of Northern European descent don’t make one of the receptors for HIV. They are resistant to infection. But this doesn’t happen for most other viruses.

Joseph wrote back:

Hmm. I thought by definition virions weren’t living organisms, yet they “adapt” to bind to living cells. Sounds like those emotional virions just can’t deal with rejection – that and our cells just aren’t as smart as we need them to be. I’m not sure if you are a Trekkie; however, it reminds me of the Borg and The Enterprise’s encounter – The Enterprise adapting to The Borg’s every frequency of their phasers, bypassing their bruteforce.

That does make sense that our cells do need that protein surface for energy; however, I never thought it would actually be the surface itself. Interesting.

I did read about that somewhere – because of the Bubonic Plague causing some genetic mutation, if I’m not mistaken.

To which I responded:

Virus particles are not alive – but once they infect a living cell they can evolve.

Both cells and viruses are smart – they both have managed to be around for a long time. We have great immune systems; virus infected cells can evolve very quickly. It’s an arms race.

Correct, one idea is that the mutation conferring resistance to HIV was acquired in the Plague, but that’s hard to prove.

The mutation we are discussing is of course ccr5delta32, which confers resistance to infection with HIV-1 (the illustration shows the HIV-1 glycoprotein binding CD4 and ccr, a chemokine receptor). You can read more about ccr5delta32 here or listen to us discuss it on TWiV #278. We also talked about virus-receptor arms races on TWiV #242, and I wrote about it here.

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On episode #279 of the science show This Week in Virology, Vincent, Alan, and Kathy reveal how a retrovirus in the human genome keeps embryonic stem cells in a pluripotent state, from where they can differentiate into all cells of the body.

You can find TWiV #279 at www.twiv.tv.