On episode #33 of the science show This Week in Microbiology, Vincent, Michael, and Ivo review the requirement for segmented, filamentous bacteria for the induction of a specific type of helper T cell in the gut.

You can find TWiM #33 at microbeworld.org/twim.

On episode #183 of the science show This Week in Virology, Connor Bamford joins the TWiV team to discuss bats as hosts for major mammalian paramyxoviruses.

You can find TWiV #183 at www.twiv.tv.

On episode #182 of the science show This Week in Virology, Michael Imperiale joins the TWiV crew to discuss the recently published influenza H5N1 transmission paper and how it was viewed by the NSABB.

You can find TWiV #182 at www.twiv.tv.

On episode #32 of the science show This Week in Microbiology, Vincent, Elio and Michael speak with Rosie Redfield about her evidence that a bacterium cannot grow on arsenic instead of phosphorus.

If you only listen to one episode of TWiM all year, make it this one – Rosie is terrific!

You can find TWiM #32 at microbeworld.org/twim.

One of two papers on avian influenza H5N1 virus that caused such a furor in the past six months was published today in the journal Nature. I have read it, and I can assure you that the results do not enable the construction of a deadly biological weapon. Instead, they illuminate important requirements for the airborne transmission of influenza viruses among ferrets. Failure to publish this work would have compromised our understanding of influenza viral transmission.

The paper from Kawaoka’s group focuses on the viral hemagglutinin (HA) protein, an important determinant of whether influenza viruses can infect birds or mammals. In the image, the HA is shown as blue ‘spikes’ on the virion surface; a single HA molecule is shown at right. Avian influenza viruses prefer to attach to cells via a specific form of sialic acid that differs from the form bound by mammalian influenza viruses. This difference in receptor preference is one reason why avian influenza viruses do not transmit among mammals.

Kawaoka’s group used a random mutagenesis and selection approach to identify amino acid changes in the avian H5 HA protein that allow it to bind human receptors. These changes are located around the sialic acid binding pocket in the HA head (figure). Some of the amino acid changes were previously known, but there are also some new ones reported, expanding our understanding of how the HA binds sialic acids. Some of the HA amino acid changes allow virus binding to ciliated epithelial cells of the human respiratory tract (wild type H5 HA cannot). All of this is important new information.

The H5 HA genes with these amino acid changes were then substituted for the HA gene in a 2009 H1N1 pandemic virus, and this reassortant virus was inoculated intranasally into ferrets. The viruses did not replicate well in the ferret trachea, but viruses recovered from the animals contained a new change in the HA protein that improves replication. This change (asparagine to aspartic acid at amino acid 158) is known to prevent attachment of a sugar group to the HA and enhance binding to human receptors. Viruses with this change probably have a replicative advantage in ferrets.

A reassortant virus with HA amino acid changes N158D/N224K/Q226L transmitted through the air to 2 of 6 ferrets. Viruses recovered from one of the animals contained a new change in the HA protein, T318I. A virus with four amino acid changes in the H5 HA (N158D/N224K/Q226L/T318I) replicates well in ferrets and transmits efficiently, although the infection is not lethal.

Even more interesting are the results of experiments to understand how these HA amino acid changes affect viral transmission. The N224K/Q226L amino acid changes that shift the HA from avian to human receptor specificity reduce the stability of the HA protein. The N158D and T318I changes, which were selected in ferrets, restore stability of the HA.

There are three key questions concerning this work that must be answered.

Would an H5N1 virus with the changes N158D/N224K/Q226L/T318I transmit among humans? Probably not. The virus tested by the authors derived 7 of 8 RNA segments from a human H1N1 strain, which is well adapted for human transmission. It is likely that changes in other avian influenza viral proteins would be needed for human transmission. It might also be that entirely different changes in the H5 HA are required for transmission in humans compared with ferrets.

Is this information useful for the surveillance of circulating H5N1 strains; specifically, would the emergence of these HA changes signify a virus with pandemic potential? I don’t believe so. These are mutations that enhance the transmission of H5 viruses in ferrets, and their effect in humans is unknown. Ferret transmission experiments are not meant to be predictive of what might occur in humans.

If these results are not predictive of what might happen in humans, why were these experiments done? (to paraphrase Laurie Garret at the New York Academy of Sciences Meeting on Dual Use Research). A substantial portion of this work goes far beyond surveillance of H5N1 strains: it provides a mechanistic framework for understanding what regulates airborne transmission of avian H5 influenza viruses. In the Kawaoka study, amino acid changes that improve the stability of the HA protein were selected for during replication and transmission of the H5 viruses in ferrets. In other words, stability of the HA protein is an important property that allows efficient airborne transmission among ferrets. Additional experiments can now be designed to extend this idea. If such stabilizing changes can be shown to be important for transmission of human strains, then they might be a valuable marker of influenza transmission.

The Kawaoka paper is a significant piece of work that substantially advances our understanding of what viral properties control airborne transmission of influenza viruses. To view it as enabling construction of a bioweapon is highly speculative and fundamentally incorrect.

M. Imai, T. Watanabe, M. Hatta, S.C. Das, M. Ozawa, K. Shinya, G. Zhone, A. Hanson, H. Katsura, S. Watanabe, C. Li, E. Kawakami, S. Yamada, M. Kiso, Y. Suzuki, E.A. Maher, G. Neumann, Y. Kawaoka. 2012. Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets.   doi: 10.1038/nature10831.

Nearly four months ago I stood at the front of a crowded classroom at Columbia University and began teaching the third year of my undergraduate virology course. Twice a week we discussed the basic principles of virology, including how virions are built, how they replicate, and how they cause disease. Yesterday was the 26th and last lecture in the course, entitled “H5N1″. In this lecture we covered the recent controversy over the publication of results on adapting avian influenza H5N1 viruses to transmit by the airborne route among ferrets. Fittingly, one of the two papers in question will be published tomorrow.

Each lecture in my virology course has been recorded as a videocast and is available at the course website, at iTunes University, or on Vimeo. Eighty-seven Columbia University undergraduates registered for the course in 2012, but over 14,000 individuals have subscribed to virology W3310 through iTunes University. I believe that it is important that the general public understand as much as possible about viruses, so they can participate in the debate about issues that impact them, such as XMRV or H5N1. It is my goal to be Earth’s virology professor.

I am sure that the students were perplexed when I took their photo before the first lecture. Little did they know that they were about to take a very different science course, one taught by a professor who uses social media (blogs, podcasts, twitter) to teach the subject both in and out of the classroom. As one student wrote to me yesterday:

I wish that every professor I had had such passion and energy and a TWiV-like blog/show so I could be updated on all the big science gossip/news to complement my in-class knowledge! I can’t recount how many times I told my non-science friends about TWiV as an exhibit to prove that science is cool and important. Thank you for being passionate scientists that made me want to study science (and be super nerdy but connected to the world) in the first place.

I would like to thank all the students of virology in and out of the classroom for their enthusiasm and their willingness to learn a complex subject. Virology will be offered again in the spring of 2013, and you can be reassured that it will be different. My course, like viruses, is continually evolving.

On episode #181 of the science show This Week in Virology, Vincent, Rich, and Kathy discuss Cotia virus, a new poxvirus, Orf virus infections associated with handling goats and lamb, and the innate immune response to prions.

You can find TWiV #181 at www.twiv.tv.

Science magazine will be conducting a live chat on whether some scientific research is too dangerous to publish, and how governments are getting involved in regulating such studies. It will be moderated by Science writer David Malakoff and will include Gregory Viglianti of Boston University School of Medicine.

The live chat begins at 3 PM EST on Thursday, 26 April at this link.

A dairy cow in California is the fourth known American case of mad cow disease, which is caused by prions, infectious agents composed only of protein (the story hit the press the day after my lecture on this type of illness). Unlike viruses, prions have no nucleic acid and no protective coat. But virologists know all about them because, as Stanley Prusiner once said, there was a time when only virologists believed that they existed.

Prions are found in mammals and in fungi, but only in mammals are they infectious and pathogenic. All mammals make normal forms of the prion protein (PrP^c) which is found in many tissues including the nervous system. The pathogenic form, called PrP^Sc, is a structurally altered form of PrP^c. The PrP^Sc protein, named after the first prion disease studied, scrapie in sheep, causes PrP^c to undergo a structural transformation to the pathogenic form. The PrP^Sc protein becomes deposited in amyloid fibrils in the brain, leading to neurodegenerative diseases known as transmissible spongiform encephalopathies (TSE), after the sponge-like appearance of the brain observed in afflicted animals (image).

There are three different ways to acquire a TSE. One is by infection: a human consumes meat that contains PrP^Sc, or receives a corneal transplant from a donor with an undiagnosed TSE . The PrP^Sc proteins make their way to the brain where they cause the host’s PrP^c to misfold and become the pathogenic PrP^Sc. The more PrP^Sc that is made, the more the normal PrP^c is converted to the pathogenic form. After an incubation period of many years, the host develops an invariably fatal neurodegenerative disease characterized by dementia in humans. There is also a familial form, in which mutations in the gene encoding PrP^c are inherited; these cause the PrP^c protein to misfold to form the pathogenic form. In the sporadic form PrP^c spontaneously converts to PrP^Sc without any known mutation or infection.

TSEs occur in different forms with varied symptoms and pathology. There are TSEs of humans (Creutzfeld-Jacob disease, fatal familial insomnia, Gerstmann-­Sträussler syndrome, Kuru) cows (bovine spongiform encephalopathy or mad cow disease), sheep and goats (scrapie), deer, elk, and moose (chronic wasting disease), and of a variety of other mammals.

This brings us back to the mad American cow, the first in the US since 2006. It died on a dairy farm and was tested for BSE as are 40,00o other cows each year in this country. The reason why this is big news is that back in the 1990s there was an outbreak of human TSE in the United Kingdom caused by consuming beef from animals with BSE. The cows acquired BSE by being fed processed animal byproducts as protein supplements, which unknowingly contained pathogenic prions. Bt the time the disease was detected in cows, contaminated meat had already entered the human food chain. Cows are routinely tested for BSE precisely to avoid a similar outbreak of human TSE.

The dead cow apparently had atypical BSE – that is, it was not a consequence of eating contaminated meat and it was not an inherited disease. Atypical BSE is caused by strains of prions distinct from other forms. This is good news because it means that the feed that the cow was receiving was not contaminated with pathogenic prions. Furthermore, the cow was not destined for meat production; it was a dairy cow that had died and was selected for random sampling.

Could the milk produced by this cow and consumed by humans pose a risk for transmission of a TSE to humans? It is known that ewes with scrapie shed infectious and pathogenic prions in their milk. However cows with BSE have  much less PrP^Sc accumulation in peripheral tissues, and in particular lymphoid tissues which include the mammary glands. It seems unlikely that cow milk contains prions, but it is a question worth revisiting. Pathogenic prions are highly resistant to heat, ultraviolet irradiation and other extreme conditions, so would certainly survive the pasteurization process.

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 www.twiv.tv.

When my laboratory discovered the cell receptor for poliovirus in 1989, many new research directions were suddenly revealed – such as creating a mouse model for poliomyelitis. One application we did not think of was to use the receptor to screen samples of drinking water for the presence of viruses.

Contamination of the water supply with fecal material can lead to the presence of enteric viruses, which constitute a public health risk. A variety of methods have been developed to screen for viruses in water samples using cell culture or nucleic acid detection techniques. Because the numbers of human viruses in water samples are low, a concentration step must usually be included. These are typically laborious and costly, and innovative improvements are needed. Enter CD155, the cellular receptor for poliovirus, which the authors used as a model for developing a new way to concentrate viruses from water samples.

Viral receptors, which are present on the surface of susceptible cells, are very efficient at capturing viral particles. Why not put these receptors on the surface of bacteria, where they can bind to viruses? Concentrating the viruses would then be a simple matter of centrifuging the bacteria from the water sample. This concept was tested by using poliovirus and the poliovirus receptor.

For this method to work, the viral receptor protein must be present on the surface of bacteria (Figure). To accomplish this goal, an artificial gene was made which codes for the poliovirus receptor protein fused to the ice nucleation protein (INP) gene. This protein is normally present on the surface of the bacterium Pseudomonas syringae.

When the PVR-INP gene was expressed in E. coli, the fusion protein was located to the surface of the bacteria, where it could bind poliovirus. The recovery efficiency was then tested by adding poliovirus to tap water, saline, and samples from several local rivers. The engineered bacteria were added to the poliovirus-laced waters and mixed for 20-60 minutes. The bacteria were then removed by low speed centrifugation, and the viral titers in the cell and in the liquid sample were determined by plaque assay. The recovery of infectious virus ranged from 99% (saline samples) to 75% (river water).

These findings demonstrate that recombinant bacterial cells can be used to capture virus particles in different types of water samples. Compared with other water concentration methods, centrifugation is inexpensive and easy. Whether or not this assay is sensitive enough to detect low levels of viruses in drinking water and other samples must still be determined.

In a way it is fitting that bacteria have been used to capture poliovirus. After all, poliovirus initially replicates in the gastrointestinal tract, where the microbial flora (including E. coli) helps the virus invade the host.

Abbaszadegan M, Alum A, Abbaszadegan H, Stout V. 2011. Cell surface display of poliovirus receptor on Escherichia coli, a novel method for concentrating viral particles in water. Appl Envir Micro 77:5141–5148.

On episode #31 of the science show This Week in Microbiology, Vincent, Jo, and Michael discuss an archetypal protein transport system in bacterial outer membranes, and evidence that gut microbial enterotypes might not fall into defined groups.

You can find TWiM at microbeworld.org/twim.

On episode #179 of the science show This Week in Virology, Gertrud joins the TWiVoners to review how dengue virus infection of mosquitoes alters blood feeding behavior, and gene therapy as practiced by parasitoid wasps.

You can find TWiV #179 at www.twiv.tv.

Influenza H5N1 virus frightens many because of the widely quoted case fatality ratio of >50%, which is based on the number of deaths among the fewer than 600 cases confirmed by the World Health Organization. Such fear is misguided, because it is likely that the fatality ratio is far lower. For example, studies of >7,000 healthy individuals have revealed that about 0.5% of them carry antibodies to the virus in their blood, indicating that mild or asymptomatic infections do occur. T-lymphocytes that recognize influenza H5N1 virus have now been detected in a high-risk cohort of individuals in Vietnam, providing additional evidence for asymptomatic human infections.

Viral infection of a healthy host usually leads to the production of both antibodies and lymphocytes. Antibodies generally bind to virus particles in the blood and at mucosal surfaces, blocking the spread of infection. In contrast, T-cells recognize and kill infected cells. The presence of specific antibodies has historically been used as an indicator of viral infection, partly due to the simplicity of the assay. In practice, dilutions of patient sera can be mixed with infectious virus to measure its ability to block infection (virus neutralization or hemaggultination-inhibition), or used to detect antibodies to viral proteins (ELISA or western blot).

Another option for assessing previous viral infection is to determine whether virus-specific T-cells are present. In the past this type of assay has been difficult to carry out with large numbers of samples, but technical advances have now made it possible to do population screens for T-cell responses. In the current study, peripheral blood mononuclear cells (which contain T-lymphocytes and other blood cells) were obtained from patients and placed in culture. Next, pools of overlapping peptides that span most of the influenza viral proteins were added to the cells. These peptides were derived from influenza H5N1, H3N2, and H1N1 proteins. When T-cells recognize a peptide (via the T-cell receptor that recognizes the peptide presented by another cell type; see figure), the cells respond by producing interferon gamma, which can be readily measured.

The Vietnamese patients used for this study were part of a rural community where human and avian infections with influenza H5N1 had been previously documented. Twenty-four of of 747 individuals had evidence for the presence of T-cells that recognize peptides from the H5 HA more strongly than peptides from H3 or H1 HA. Another 111 samples had T-cells that react with H5, H1, and H3 peptides. If all positive patient samples (those lead to production of interferon gamma) are included, then one can conclude that 20% of the patients respond to H5 peptides. Control samples (n=271) were obtained from two individuals in Vietnam and the United Kingdom who are not believed to have been exposed to H5N1 virus. None of these had H5N1-specific T-cell responses.

Curiously, only four subjects had both antibody and T-cell responses to H5N1 virus. The timing of sample acquisition with respect to infection is likely to be important for detecting responses. For example, antibody to H5N1 virus may not be detected earlier than 3 weeks after onset of disease, and T-cell responses may wane with time. It is also possible that abortive H5n1 infections in humans may lead to production of T-cells but not antibodies, as is seen in some individuals infected with HIV-1.

On episode #178 of the science show This Week in Virology, the TWiValians meet up with Tyler Sharp for a discussion on the Epidemic Intelligence Service and controlling dengue.

You can find TWiV #178 at www.twiv.tv.

On episode #169 of This Week in Virology we had a good discussion about how to read a scientific paper. Many individuals have asked about making this into a separate audio file, so here it is.

Click the arrow above to play, or right-click this link to download our thoughts on how to read a scientific paper (22 MB .mp3, 30 minutes).

Epidemiologist Michael Walsh has shared a PowerPoint presentation on this topic (482 KB PowerPoint file).

On episode #30 of the science show This Week in Microbiology, Vincent, Elio, and Michael review how a toxin from Burkholderia pseudomallei inhibits protein synthesis, and the role of the gut microbiome in modulating insulin resistance in mice lacking an innate immune sensor.

You can find TWiM #30 at microbeworld.org/twim.

This past February I was interviewed by the Australian Broadcasting Company on the topic of the Fouchier and Kawaoka experiments on avian influenza virus H5N1. The video, Building the Perfect Bug, has been released by Journeyman Pictures and includes interviews with S.T. Lai, Laurie Garrett, Michael Osterholm, and Ron Fouchier (transcript available). It is far too alarmist for my taste, but both sides of the issue are represented.

The video includes sequences of me working in the cell culture laboratory. Note that I did wear a tie for my interview while Michael Osterholm did not.

On episode #38 of the science show This Week in Parasitism, Vincent and Dickson tackle the backlog of listener email, then consider the life cycle and pathogenesis of Trichomonas vaginalis, the flagellated protozoan transmitted by sexual contact.

You can find TWiP #38 at microbeworld.org/twip.

On episode #177 of the science show This Week in Virology, Vincent, Connor Bamford, Wendy Barclay, and Ron Fouchier discussed avian influenza H5N1 transmission experiments in ferrets and novel bunyaviruses at the 2012 Spring Conference of the Society for General Microbiology in Dublin, Ireland.

You can find TWiV #177 at www.twiv.tv.

The National Science Advisory Board for Biosecurity (NSABB) has re-examined two manuscripts on the transmissibility of influenza H5N1 virus in ferrets:

After careful deliberation, the NSABB unanimously recommended that this revised Kawaoka manuscript should be communicated in full. The NSABB also recommended, in a 12 to 6 decision, the communication of the data, methods, and conclusions presented in this revised Fouchier manuscript.

The NSABB reached this decision using ‘analytical tools that it previously developed for considering the risks and benefits associated with the communication of dual use research of concern.

Apparently information communicated in revised versions of the Fouchier and Kawaoka manuscripts changed the Board’s risk/benefit calculation:

The data described in the revised manuscripts do not appear to provide information that would immediately enable misuse of the research in ways that would endanger public health or national security.

New evidence has emerged that underscores the fact that understanding specific mutations may improve international surveillance and public health and safety.

This decision (full text here) is welcome, although I wonder how the manuscripts have been ‘revised’ – were data added or removed? Furthermore, why does the NSABB now feel that the results do not endanger public health, and can be used to improve international surveillance? These arguments have been made previously but the NSABB discounted them.

I look forward to publication of the Fouchier and Kawaoka findings and a comprehensive discussion of how they influence influenza H5N1 transmission in ferrets.

Update: According to the New York Times, the chair of the NSABB said “the new decision was not a reversal, because the revised manuscripts were so different from the originals. Had these versions been presented originally, the board would not have recommended withholding any details”.

Did the authors remove data from the manuscripts, or just clarify them?

Update 2. According to Kawaoka, quoted in the The Chronicle of Higher Education, the revisions of his manuscript “provided a more in-depth explanation of the significance of the findings to public health and a description of the laboratory biosafety and biosecurity.” His paper, he added, would contain descriptions of all the mutations that enhanced transmission of the virus, the very data that initially concerned the board.” Furthermore, Ron Fouchier wrote to me in an email that “the manuscripts will indeed be published in full.” All this is very good news.

I just returned from Dublin where I was honored to receive the Peter Wildy Prize for Microbiology Education from the Society for General Microbiology. This prize is awarded annually for an outstanding contribution to microbiology education, including university teaching, education of the general public, school pupils or professional groups.

Below is a video of my acceptance talk. Thanks to Chris Condayan of ASM for the excellent split-screen recording.

While at the SGM meeting in Dublin I recorded TWiV 177 with Connor Bamford, Wendy Barclay, Richard Elliott and Ron Fouchier. Audio and video will be posted on 1 April 2012.

On episode #176 of the podcast This Week in Virology, Vincent, Alan, and Rich answer listener email about MS, CFS, EBV, B cells, virii, influenza B, scientific papers, and more.

You can find TWiV #176 at www.twiv.tv.

Some viruses that infect bacteria (bacteriophages) deliver their DNA into the host cell with an amazing injection machine. The tailed bacteriophages (such as T4, illustrated) store their DNA in a capsid attached to a long tail tube that is surrounded by a sheath. At the bottom of the tube is a baseplate with a spike in the center. When the baseplate contacts the host cell, the sheath contracts, driving the spike into the cell membrane. The viral DNA travels down the tube and enters the cell through the opening produced by the spike. The structure of the spike has now been determined, providing insight into how it makes a hole in the cell membrane.

Structures of the spike from P2, a well studied virus of E. coli, and the choleraphage phi92 were determined.  The spikes are built from three copies of a single protein (trimers). The trimers are indeed shaped like spikes: they are wider at one end and taper to a rather sharp tip (figure at right). The bulk of the spike is made up of alternating beta-strands which form a corkscrew-like beta-helix. The sharp tip is composed of three beta-hairpins which the authors say “come together like petals in a flower bud”.

An interesting feature on the interior of the spike tip are three pairs of histidine residues that hold a single iron atom (figure at left). The authors believe that the iron helps the trimers form by keeping the protein chains in register, and also provides increased strength to the tip. The latter would be important as it pierces the cell membrane. This idea could be tested by changing one or more histidines to another amino acid so that iron cannot be held in the tip.

To verify that these structures are those of the spike that is attached to the baseplate, the authors solved the structure of the phage particle by cryo-electron microscopy and image reconstruction. The image clearly shows the spike protruding from the base plate (figure below). The structures of the spike proteins solved by X-ray crystallography could then be computationally fitted in the correct location in the cryo-EM image of the baseplate.

These structures support the idea that the spike is a rigid needle that pierces the bacterial membrane and forms a channel through which the DNA can pass. An interesting question is how the DNA gets past the spike, which plugs the end of the tail tube. The authors believe that the spike is loosely attached to the tube and might be easily dissociated once it passes through the cell membrane. The spike of phage T4 can be dissociated at low pH, a condition that is found in the periplasm, the space between the inner and outer bacterial membranes.

There are distinct signatures of the spike structure that can be identified in proteins of other contractile injection systems, including diverse bacteriophages. They can also be found in bacterial type VI secretion systems, which are membrane complexes used to transport proteins outside of the cell. Once evolution builds a useful machine, it is often put to many diverse uses.

 
Browning, C., Shneider, M., Bowman, V., Schwarzer, D., & Leiman, P. (2012). Phage Pierces the Host Cell Membrane with the Iron-Loaded Spike Structure, 20 (2), 326-339 DOI: 10.1016/j.str.2011.12.009

On episode #29 of the podcast This Week in Microbiology, Vincent and Stanley review how a phage pierces the cell membrane with an iron-loaded spike, and two programmed cell death systems in E. coli.

You can find TWiM #29 at microbeworld.org/twim.

Join us for a live-streaming episode of This Week in Virology from the Society for General Microbiology 2012 Spring Conference in Dublin, Ireland. My guests for this special episode are Connor Bamford, Wendy Barclay, Richard Elliott, and Ron Fouchier.  Watch the live stream below which starts on Monday, 26 March at 3:30 PM GMT (10:30 AM EST | 7:30 AM PST). If you have questions during the broadcast you can tweet them using the #TWiV hash tag.

You can use www.everytimezone.com to calculate when the live streams will start in your area.

If you are using an iOS device, you will not be able to see the live stream below as it is flash encoded.

On episode #175 of the podcast This Week in Virology, Vincent, Alan, and Matt discuss herpes simplex encephalitis in children with innate immune deficiency, and the local response to microneedle-based influenza skin immunization.

You can find TWiV #175 at www.twiv.tv.

A consequence of the recent warm weather in the northeastern United States is the emergence of crocuses, an event that I documented at the TWiV Facebook page. A reader replied that it reminded her of the highly valued tulips with beautiful variegations produced by viruses.

In 17th-century Holland patterned tulips such as the Semper Augustus (image) were of enormous value, with single bulbs selling for 3000 guilders or more (about $1600 US today). The intricate lines and flame-like streaks produced stunning effects. We now know that these colorful patterns are caused by infection with potyviruses, which are filamentous plant viruses with positive-strand RNA genomes. The specific viruses involved are tulip-breaking virus, tulip top-breaking virus, tulip bandbreaking virus, and Rembrandt tulip-breaking virus. Lilies may also be patterned by infection with Lily mottle virus. These viruses infect the bulb and cause the single color to break, leading to bars, stripes, streaks, featherings or flame-like effects of different colors on the petals. These effects are caused by altered distribution of pigments in the petal caused by virus replication.

Unfortunately, infection with tulip-breaking viruses is not benign: with successive generations the bulb shrinks until it can no longer flower. For this reason most of the lines of broken tulips, including Semper Augustus, no longer exist. These viruses still circulate globally, transmitted by aphids. Because infection can cause costly damage to tulips, precautions must be taken to minimize spread. Contemporary variegated tulips such as Rem’s Sensation are produced by breeding, not virus infection.

It is spring break for students at Columbia University, which means that my annual virology course is one lecture past the halfway point. The first eleven lectures addressed basic aspects of viral replication in cell culture, including virus entry into cells, genome replication, and assembly. From this point onwards we will be discussing viral infection of a host, including pathogenesis, immunity, immunization, antivirals, and evolution.

All my virology lectures are available as videocasts (slides and audio) either at the course website, or at the new iTunes University.

On episode #174 of the podcast This Week in Virology, Vincent, Alan, and Rich consider whether pet dogs might transmit human noroviruses, and an RNA virus microRNA that might be involved in oncogenesis.

You can find TWiV #174 at www.twiv.tv.

On episode #173 of the podcast This Week in Virology, the TWiVites discuss seroevidence for human infection with avian influenza H5N1, and the discovery of a new influenza virus in Guatemalan bats.

You can find TWiV #173 at www.twiv.tv.

Ron Fouchier has discussed his influenza H5N1 transmission experiments in ferrets at an ASM Biodefense Conference, clarifying several assumptions about the transmissibility of the virus in this animal model.

Two different influenza H5N1 strains were used for Fouchier’s experiments: a wild type virus, and a mutated virus (we’ll call it mutH5N1). He did not reveal the nature of the mutations in this virus but from previous reports they consist of changes introduced into the viral HA protein to allow binding to sialic acid receptors in the avian respiratory tract, and other changes selected during passage in ferrets.

Ferrets are housed in neighboring cages separated by steel grids to allow free air flow between cages. The cages are placed in a class 3 biosafety hood within a BSL3+ facility. A ferret in one cage is inoculated intranasally with virus, and then ferrets in neighboring cages are assayed for presence of virus in the respiratory tract. When ferrets are inoculated with wt H5N1 virus, viral replication ensues in the respiratory tract, but the virus is not transmitted to animals in neighboring cages. When ferrets are inoculated with mutH5N1, the virus is transmitted to 3/4 ferrets in neighboring cages. If the mutH5N1 virus is recovered from these animals and used to infect new ferrets, it is then transmitted to 2/2 ferrets in neighboring cages. The results are summarized in the following figure:

Fouchier concluded that this work identified the mutations that are needed for H5 transmission between ferrets.

Next Fouchier indicated that because the work has not yet been published, and the press has ‘picked up on it’, there are many misconceptions about what can or cannot be concluded. For example, it has been suggested that this virus would spread ‘like wildfire’ if it were to get out of his facility. He presented data indicating that this would not be the case. Although his results demonstrate aerosol transmission of H5N1 among ferrets, the assay is not quantitative, and therefore the efficiency of transmission cannot be deduced. He showed results of ferret transmission studies using the 2009 H1N1 pandemic influenza virus strain. This virus spreads to all ferrets by aerosol and replicates to high titers in the respiratory tract. In comparison, the mutH5N1 virus does not transmit to all ferrets, virus titers are lower, and shedding does not begin until later in infection. He concluded that the mutH5N1 virus does not transmit among ferrets as does a pandemic or seasonal influenza virus.

The second misconception that he addressed is that the mutH5N1 virus would be highly lethal. He showed the results of experiments demonstrating that when ferrets are inoculated intranasally with high doses of mutH5N1 virus, only 1/8 animals show signs of disease. In contrast, 2 of 2 ferrets developed disease when inoculated in the same way with wild type H5N1 virus. When the mutH5N1 virus is transmitted to ferrets via aerosol, none of the recipient animals develop disease. Only when the mutH5N1 virus is delivered to the lower respiratory tract of ferrets by intratracheal intubation does the virus cause disease in 6 of 6 animals.

Finally, Fouchier showed that pre-exposure of ferrets to seasonal influenza virus protects them from disease caused by H5N1 viruses. These findings are summarized on the following figure.

After this presentation Dr. Anthony Fauci, head of the National Institute of Allergy and Infectious Disease, said that “There is a gross, pervasive misunderstanding out there,” and recommended that the data be re-examined by the NSABB.

The data presented by Fouchier appear to be at odds with the conclusions of the NSABB to redact publication. They are also not consistent with statements made by Fouchier and others to Science magazine in November 2011. For example, Fouchier called mutH5N1 ”probably one of the most dangerous viruses you can make”, and Paul Keim, head of the NSABB, said ”I can’t think of another pathogenic organism that is as scary as this one.”

Update: Here is what Fouchier said about his work at the Malta meeting in September 2011, as reported by New Scientist. The article begins with the statement:

…five mutations in just two genes have allowed the virus to spread between mammals in the lab. What’s more, the virus is just as lethal despite the mutations.

Fouchier is quoted as saying ”The virus is transmitted as efficiently as seasonal flu.” This is in direct contrast to what he reported at the ASMBiodefense meeting.

In describing the passage of H5N1 in ferrets, the writer concludes:

The tenth round of ferrets shed an H5N1 strain that spread to ferrets in separate cages – and killed them.

Again this is in direct contrast to what Fouchier reported this past week.

I do not understand the difference between what Fouchier said in Malta in 2011 and in Washington, DC in February 2012. However, there is one way to explain the apparent paradox, which derives from the following statment from the New Scientist article:

The process yielded viruses with many new mutations, but two were in all of them. Those plus the three added deliberately “suggest that as few as five are required to make the virus airborne”, says Fouchier. He will now test H5N1 made with only those five.

Perhaps the results that Fouchier reported in Washington, DC are from experiments using H5N1 virus with only those five mutations.

On episode #172 of the podcast This Week in Virology, Vincent and Kathy discuss how a virus may cause disease distant from its replication site, then review a day in the life of a senior microbiology professor.

You can find TWiV #172 at www.twiv.tv.

The fatality rate for human infections with avian influenza H5N1 is widely quoted at >50%, based on the number of deaths among the fewer than 600 cases confirmed by the World Health Organization. Wang, Parides, and Palese suggest that this number is an overestimate:

…the stringent criteria for confirmation of a human case of H5N1 by WHO does not account for a majority of infections, but rather, the select few hospitalized cases that are more likely to be severe and result  in poor clinical outcome.

To address this problem, the authors summarized the results of serological surveys in which human sera were examined for the presence of antibodies to influenza H5N1 virus. Because antibodies are part of our immune defenses, they are a good indicator of a previous infection.

The authors searched the scientific literature and identified 20 studies in which human sera were examined for the presence of H5N1 antibodies according to WHO guidelines (a 4-fold or greater increase in neutralizing antibody titer in paired acute and convalescent sera, with the convalescent serum having a titer of ≥1:80, or an antibody titer of ≥1:80 in a single serum collected at day 14 or later after onset of symptoms and a positive result using a different serological assay).

Studies that used the WHO criteria included 7,304 study participants. Rates of seropositivity were from 0 – 5.3%, with one study reporting 11.7% positivity. The meta-analysis yielded a seropositivity rate of 1.2% (95% confidence interval 0.6% – 2.1%). When only poultry workers were considered, the seropositivity rate was 1.4%.

Other studies were separately analyzed that did not utilize WHO guidelines; these included 6,774 participants and yielded a seropositivity rate of 1.9% (95% confidence interval 0.5 – 3.4%).

A total of 12,677 study participants from 20 studies were included in this meta-analysis, of which 1-2% had evidence for prior H5N1 infection. The authors conclude:

…avian H5N1 viruses can cause a rate of mild or subclinical infections in humans that is not currently accounted for and thus, the true fatality rate for H5N1 influenza viruses is likely to be less than the frequently reported rate of more than 50%.

It seems very clear that standardized, large scale studies are needed to determine the real number of human H5N1 infections. This information is critical for assessing the actual threat of H5N1 influenza for humans.

For the second time in a week I note the passing of an important virologist. Renato Dulbecco, together with David Baltimore and Howard Temin, received the 1975 Nobel Prize in Physiology or Medicine for discoveries about how tumor viruses interact with the genetic material of the cell. Dulbecco also devised my favorite virological method, the plaque assay, for determining the virus titer, the number of animal viruses in a sample.

Since the early 1920s bacteriologists had used the plaque assay to quantify the number of infectious bacteriophages (viruses that infect bacteria). Dulbecco noted in 1952 that “research on the growth characteristics and genetic properties of animal viruses has stood greatly in need of improved quantitative techniques, such as those used in the related field of bacteriophage studies.” One limiting factor was the development of suitable animal cell cultures that could be used to determine viral titer. By the 1950s the techniques for reliably producing and propagating human cell cultures were developed, and in 1951 the first immortal human cell line, HeLa, was isolated. Dulbecco took advantage of these advances and showed in 1952 that western equine encephalitis virus formed plaques on monolayers of chicken embryo fibroblasts (figure). Dulbecco also made the important observation that one virus particle is sufficient to produce one plaque. He drew this conclusion from his observation of a linear dependence of the number of plaques on virus concentration. This seminal advance made possible the application of genetic techniques to the study of animal viruses.

Dulbecco’s work on tumor viruses was focused on polyomaviruses – small DNA-containing viruses such as murine polyomavirus and SV40. He found that cells from the natural host of the virus – mice for polyomavirus and monkeys for SV40 – were killed as the viruses replicated and produced new viral progeny. However, these viruses did not replicate in or kill cells from other animals. For example, when hamster cells were infected with murine polyomavirus, no viral replication took place, the cells survived, and a few rare cell were transformed  - their growth properties in culture were altered and they induced tumors when injected into hamsters. Dulbecco later found that the polyomaviral DNA is a circular, double-stranded molecule; and that in non-permissive cells (in which the virus does not replicate) the viral DNA became integrated into the host cell chromosome. He also suspected that a viral protein called T (for tumor) antigen was a key to cell transformation.

Today we understand why polyomaviruses transform cells in which they do not replicate: infection does not kill these cells, and the rare transformed cells contain only viral DNA encoding T antigen. This protein is needed for viral replication in permissive cells because it drives cell proliferation, activating cellular DNA replication systems that are required for producing more viral DNA. In a non-permissive cell, T antigen drives the cell to divide endlessly, immortalizing it and allowing the accumulation of mutations in the cell genome that make the cells tumorigenic.

While the details of how DNA tumor viruses transform cells were being elucidated, other investigators were attempting to understand how another class of viruses – with RNA genomes – had similar effects on cells. In 1951 a young scientist named Howard Temin joined Dulbecco’s laboratory to study how Rous sarcoma virus (RSV) caused tumors. This virus had been discovered by Peyton Rous in 1911, but would only cause tumors in chickens, limiting progress. In Dulbecco’s laboratory, Temin found that RSV induced transformation of cultured chicken embryo fibroblasts – the same types of cells that were being used to develop the plaque assay for animal viruses. Temin took this transformation assay to his own laboratory, where he reasoned that a DNA copy of the RSV viral genome must be integrated into the chromosome of transformed cells. This led him to discover the enzyme reverse transcriptase in RSV particles, which produces a DNA copy of the viral RNA.

By embracing a new technology for the study of animal viruses – cell culture – Dulbecco set the study of both DNA and RNA tumor viruses on a path that would lead to understanding viral transformation, an achievement recognized by the 1975 Nobel Prize.

Dulbecco, R. (1952). Production of Plaques in Monolayer Tissue Cultures by Single Particles of an Animal Virus Proceedings of the National Academy of Sciences, 38 (8), 747-752 DOI: 10.1073/pnas.38.8.747

On episode #171 of the podcast This Week in Virology, Matt Frieman joins Vincent, Alan, and Dickson to review virus production in single cells and single virion genomics.

You can find TWiV #171 at www.twiv.tv.

Dr. Bruce Alberts, editor of Science magazine, said that the journal will publish the full version of the Fouchier H5N1 influenza virus paper if mechanisms are not developed to ensure circulation of the information to scientists. Alberts made his comments at the American Association for the Advancement of Science meeting in Vancouver:

Our position is that, in the absence of any mechanism to get the information to those scientists and health officials who need to know and need to protect their populations and to design new treatments and vaccines, our default position is that we have to publish in compete form.

Pallab Ghosh of BBC News, writes that Alberts ’says it is important to get the research out quickly to scientists and health officials monitoring the virus.’

Update: WHO has decided to delay the decision on whether to publish the H5N1 data:

The Geneva meeting of 22 scientists and journal representatives agreed that publishing only parts of the research would not be helpful, because they would not give the full context of a complete paper.

Update 2: The New York Times reports that the H5N1 papers will be published in a few months, “to give researchers and officials an opportunity to provide better information to the public about the research and its importance, and would also give safety experts a chance to assess the conditions in which the work is being done.”

Apparently the decision was not unanimous, and it was opposed by the US.

The other important news is that the issue of the H5N1 fatality rate has finally made it to the front page:

The experiments involve a type of bird flu virus known as H5N1. Of about 600 known cases, more than half have been fatal. The exact death rate is not known, however, because some deaths may go uncounted and mild cases may go undiagnosed.

Norton Zinder made two important discoveries in the field of virology. While a Ph.D. student with Joshua Lederberg at the University of Wisconsin-Madison he found that viruses of bacteria (bacteriophages) could move genes from one host to another, a process called transduction. Later in his own laboratory at The Rockefeller University he isolated the first bacteriophages that contain RNA as genetic material. These were seminal findings in the growing field of molecular biology.

By the 1950s it was well known that different strains of the bacterium Escherichia coli could exchange genes in a process called recombination. Zinder wanted to know if other bacteria could also exchange genes in a similar manner, and therefore began to study Salmonella typhimurium. The strains that Zinder used for his experiments were lysogens: their chromosomal DNA contained integrated copies of the DNA genomes of bacteriophages. Zinder readily detected genetic exchange in Salmonella, but suspected that the latent phages might play a role. To test this idea, he grew Salmonella in tubes that were connected by a fine filter that allowed passage of viruses but not bacteria between the two cultures. The results showed that a filterable agent, or virus, could mediate the exchange of genetic material between bacterial strains; direct contact between bacteria was not necessary. The authors coined transduction to describe this process. We now understand that transduction occurs because bacteriophages may incorporate bacterial DNA into the viral particle. Transduction remains a common tool to stably introduce a foreign gene into a host cell.

Zinder describes the discovery of RNA-containing bacteriophages, which took place after he had moved to The Rockefeller University, in the Preface to RNA Phages:

In the late fifties, Tim Loeb, a new graduate student at The Rockefeller University, came into my laboratory and asked whether I thought it was possible that there were male-specific bacteriophages for E. coli. I….quickly responded in the affirmative and off he went to a raw sewage plant in New York City. …f2, the second isolate, was chosen for further study. Little did we think at the time that a whole new area of study was in the offing….

The first two bacteriophages that Loeb had isolated from New York City sewage were called f1 and f2. During purification of the phages it was clear that the genome of f1 was DNA. Chemical analyses subsequently demonstrated that the genome of f2 was RNA (later found to be positive-strand RNA). Similar phages were since isolated all over the world, and their study provided much basic information on viral replication, protein biosynthesis, and genome replication. The first genome sequence determined was in 1976 for the related RNA bacteriophage MS2.

Update: Moving eulogy by Jeffrey Ravetch in Eulogy for a brilliant mentor and teacher.

Loeb, T. (1961). A Bacteriophage Containing RNA Proceedings of the National Academy of Sciences, 47 (3), 282-289 DOI: 10.1073/pnas.47.3.282

On This Week in Virology #170, hosts Alan, Rich, and Dickson discuss Edward Jenner’s paper on cowpox vaccine, then move 200 years later to modern vaccines against norovirus, influenza H5N1, and more.

You can find TWiV #170 at www.twiv.tv

Several panelists from the recent influenza H5N1 dual-use forum at the New York Academy of Sciences spoke with Brendan Maher of Nature News to discuss their position. Participants in this video include Laurie Garrett, Michael Osterholm, Ian Lipkin, Vincent Racaniello, and Veronique Kiermer.

Update: The New York Academy of Sciences has posted video of the full two hour panel discussion.

On This Week in Virology #169, Michael Walsh and the TWiV team review epidemiology basics, including fatality ratios.

You can find TWiV #169 at www.twiv.tv.

Howard Markel, Professor of the history of medicine at the University of Michigan, in the New York Times:

The censorship of influenza research will do little to prevent its misuse by evildoers — and it may well hinder our ability to stop influenza outbreaks, whether natural or otherwise, when they do occur. In this case, censorship is too little, too late. The data generated by one of the research teams was already presented at a conference in Malta in September, where copies of the paper were distributed. But even if the data weren’t already available, the key details could likely be inferred from other information that is already available.

Dr. Markel also agrees that influenza H5N1 virus would not be a good terrorist weapon:

Even if terrorists got their hands on the new data, it’s not certain they could weaponize the virus: no one knows for certain that the virus’s transmissibility and virulence in ferrets means transmissibility and virulence in humans. In any event, the influenza virus, highly variable in its power and spread, is not an optimal terrorist weapon, not least because no one would know for sure if it was unleashed by a terrorist or natural forces.

The implications of the H5N1 story go far beyond ferrets:

In the years since the 9/11 attacks, we’ve witnessed a disturbing trend in the oversight of sensitive science. [...] Several prominent scientists, including Donald Kennedy, the former editor of Science, have publicly worried that the federal government is thwarting scientific advancement.

The action by the NSABB has propelled us full-tilt into this controversy. Their incorrect decision to censor the influenza H5N1 data not only will inhibit work on this important virus, but will have far-reaching consequences for scientific research. To this day I cannot understand why the NSABB did not more thoughtfully consider not only the data, but the future of science in coming to their decision.

Thanks to Dr. Markel for writing the Op-Ed I’ve been meaning to put together for a long time.

Related:

The NSABB speaks on influenza H5N1

H5N1 facts, not fear

Moratorium on influenza H5N1 transmission research

Palese: Don’t censor live-saving science

N.Y. Times: H5N1 ferret research should not have been done

Should we fear avian H5N1 influenza?

Ferreting out influenza H5N1

The National Science Advisory Board for Biosecurity (NSABB) has published “Adaptations of Avian Flu Virus Are a Cause for Concern”, an explanation of their recommendations with respect to influenza H5N1 research (versions at Science and Nature). It starts with the statement that advances in technology now allow manipulation of microbial genomes in ways that could be misused, leading to global harm. They define dual-use research as “research that could be used for good or bad purposes”.

The authors begin their discussion of influenza H5N1 with the usual incorrect statement about the lethality of the virus:

Highly pathogenic avian influenza A/H5N1 infection of humans has been a serious public health concern since its identification in 1997 in Asia. This virus rarely infects humans, but when it does, it causes severe disease with case fatality rates of 59%.

The reference for this information is a WHO summary of confirmed human cases of H5N1. Both WHO and NSABB ignore the serological evidence for many mild or inapparent H5N1 infections. Omitting these data leads to an overestimation of the virulence of the virus, which has apparently played a large role in the NSABB’s decision.

Next, they engage in extensive speculation:

If influenza A/H5N1 virus acquired the capacity for human-to-human spread and retained its current virulence, we could face an epidemic of substantial proportions.

The virus has been circulating since the 1990s and has not acquired the capacity for human to human spread. This doesn’t mean it never will, but the possibility seems remote. The statement ‘retaining its current virulence’ of course refers to the erroneous 59% case fatality rate. What if the fatality rate is 0.1%, like seasonal influenza?

In discussing influenza H5N1 transmission in ferrets, the NSABB notes the value of the research:

The research teams that performed this work did so in a well-intended effort to discover evolutionary routes by which avian influenza A/H5N1 viruses might adapt to humans. Such knowledge may be valuable for improving the public health response to a looming natural threat.

Many have written that the research should never have been done, and that there are no benefits for human health (New York Times, Tom Inglesby, DA Henderson). Clearly the NSABB believes otherwise.

Next the NSABB describes their consideration of risk assessment of the H5N1 ferret studies. Their conclusion:

We found the potential risk of public harm to be of unusually high magnitude. Because the NSABB found that there was significant potential for harm in fully publishing these results and that the harm exceeded the benefits of publication, we therefore recommended that the work not be fully communicated in an open forum.

But there is no description of how they reached this conclusion. What data did they consider when making this decision? What were the benefits and the potential harms, and how did they weigh them? Apparently we must take the word of the panel that they reached the right decision, even though we cannot know what information they used. To convey their decision in this manner is unacceptable and sends the message that the committee did not consider specific data during their deliberations.

They conclude:

The life sciences have reached a crossroads. The direction we choose and the process by which we arrive at this decision must be undertaken as a community and not relegated to small segments of government, the scientific community, or society.

This is precisely why the decision to redact publication should not have been made by the NSABB or any small group of individuals. I agree that this is an ‘Asilomar moment’, a time when scientists must meet to decide what types of microbial research should be regulated. This should be a discussion among a large group of scientists, not bioterrorism policy analysts.

I understand the need to regulate certain types of experiments on microbes. But when I balance the benefits and risks of the H5N1 ferret transmission experiments, it does not make sense to stamp them as dual use and restrict publication of the results. Let publication proceed and then decide how to decide on how to move forward.

Hosts: Vincent Racaniello, Dickson Despommier, Rich Condit, Alan Dove, and Welkin Johnson

Welkin joins the TWiV team for a discussion of HIV prophlaxis using vectored antibodies, and the influenza H5N1 virus studies in ferrets that were not redacted.

Click the arrow above to play, or right-click to download TWiV 168 (59 MB .mp3, 98 minutes).

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

Links for this episode:

• Vectored HIV immunoprophylaxis (Nature)
• Vectored immunoprophylaxis – the Movie (YouTube)
• Adeno-associated virus (Wikipedia)
• In vitro evolution of H5N1 towards human receptor specificity (Virology)
• Endogenous viral genes non-essential in chicken (Nature)
• Rates of HIV transmission per coital act (J Inf Dis)
• TWiV on Facebook
• Letters read on TWiV 168

Weekly Science Picks

Welkin – Virtual PI (Nature)
Dickson – Drain the Ocean
Rich – Nova: To the Moon
Alan - Robert Falcon Scott on Twitter and the Terra Nova expedition
Vincent – Hello, Mr. Chips (I, Cringely)

Listener Pick of the Week

Charlotte – And the Band Played On

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

Peter Palese and Taia Wang have written a compelling article that uses scientific facts to address the controversy over publication of research involving transmission of avian influenza H5N1 in ferrets. In response to calls in the media to destroy the viruses, curtail the research, and protect the public from frightening research, they write that “fear needs to be put to rest with solid science and not speculation”.

The authors begin with the facts: they indicate that the object of ferret-to-ferret passage of avian H5N1 influenza virus was to determine whether sustainable aerosol transmission could be achieved in this animal model. The finding that transmission in ferrets is conferred by a small number of mutations emphasizes the need for continued surveillance of H5 viruses and development of vaccines and antivirals.

Are these studies relevant to humans?

Ferrets are quite susceptible to infection with influenza viruses. However, it is not clear that all virus strains that replicate in and transmit between ferrets necessarily do so in humans. Ferrets are also more likely than humans to have disseminated, multiorgan influenza disease including neurological sequelae….one cannot directly extrapolate from the data to make predictions about humans.

Under the heading ‘fear’, they address the heart of this controversy, the notion that the fatality rate for human H5 infections is greater than 50%:

…in order for a case to be confirmed by WHO, a person must have an acute, febrile respiratory illness with known H5 exposure in the 7 days preceding and have molecular confirmation of H5 infection by a WHO approved laboratory. This definition does not allow for asymptomatic infections and essentially requires that a person actively seek medical help at a hospital that is equipped to draw samples and ship them to an approved laboratory….it seems unlikely that even a small fraction of the total number of infected cases has been accounted for under the WHO surveillance system.

They also review seroevidence in humans for H5 infections:

Of the 10 largest studies of which we are aware…eight report rates ranging from 0.2% to 5.6%….even if only a low percentage of the rural population is asymptomatically/subclinically infected, the case fatality rate that is offered by the WHO – and that is driving this controversy – is likely orders of magnitude too high.

The authors believe that selection of these papers for redaction by the National Science Advisory Board for Biosecurity appears arbitrary, considering what has been published on influenza in the past:

In 2005, the complete sequences for the 1918 pandemic influenza virus were published…in 2006, both Science and Nature published reports of specific mutations that enable the H5 viral hemagglutinin to bind human, rather than avian tissues. In 2012, a report from the CDC that bears striking resemblance, in principle, to the works by Fouchier and Kawaoka was already published in Virology: it describes mutations in an H5N1 virus that confer airborne transmissibility between ferrets.

Finally the address the question: Could the data from these two papers realistically be used to generate an H5N1 biologic weapon?

The answer is simply no.

Everyone should read this article, including anyone who is concerned about the safety of the H5N1 experiments; biosecurity analysts who do not seem to understand the underlying science; and science writers who propagate misinformation about the virus.

Hosts: Vincent Racaniello, Dickson Despommier, Rich Condit, and Alan Dove

The complete TWiVome deconstructs the movie Contagion.

Click the arrow above to play, or right-click to download TWiV 167 (53 MB .mp3, 88  minutes).

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

Links for this episode:

• R0 explained (pdf)
• Hendra and Nipah encephalitis (CDC)
• Contagion (IMdB)
• TWiV on Facebook
• Letters read on TWiV 167

Weekly Science Picks

Dickson – Guinea Pig Doctors by Jon Franklin
Rich – Learn to appreciate technology and Everythings amazing and nobodys happy (YouTube)
Alan – JD Hooker slide collection
Vincent – iTunes U app and iBooks Author

Listener Pick of the Week

Judi – Makers of Many Things by Eva March Tappan

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

In letters to Science and Nature, the authors of the controversial avian H5N1 influenza virus transmission experiments in ferrets, together with other influenza virologists, have agreed to a 60 day moratorium on transmission research:

…we have agreed on a voluntary pause of 60 days on any research involving highly pathogenic avian influenza H5N1 viruses leading to the generation of viruses that are more transmissible in mammals. In addition, no experiments with live H5N1 or H5 HA reassortant viruses already shown to be transmissible in ferrets will be conducted during this time.

They write that research will continue on assessing the “transmissibility of H5N1 influenza viruses that emerge in nature and pose a continuing threat to human health”.

This research is being halted because of the concerns that ferret-transmissible H5N1 viruses may escape from laboratories. They argue that the finding in two laboratories that viruses with a hemagglutinin (HA) protein from highly pathogenic avian H5N1 influenza viruses can become transmissible in ferrets advances our understanding of influenza transmission. Nevertheless,

We recognize that we and the rest of the scientific community need to clearly explain the benefits of this important research and the measures taken to minimize its possible risks. We propose to do so in an international forum in which the scientific community comes together to discuss and debate these issues.

I agree in principle with this decision, because the argument over this research has become increasingly polarized in recent weeks, with a distressing demarcation between those who believe the work should proceed, and those who feel it should not be done. A dialogue to identify the crucial issues and develop plans to address them, while continuing this important line of research, is certainly welcome.

I am curious to see who will participate in the proposed dialogue. I do hope it will be a balanced forum: a fair mix of microbiologists, especially those working on influenza virus, and those interested in biosecurity. As I have said before, scientists will listen to the policy analysts, but the latter must also understand the science.

Update: Alan Dove has written an honest analysis of the moratorium announcement.

Related:

Palese: Don’t censor live-saving science
N.Y. Times: H5N1 ferret research should not have been done
Should we fear avian H5N1 influenza?
A bad day for science
Ferreting out influenza H5N1

The third annual installment of my virology course at Columbia University, Biology W3310, has begun. This course, which I taught for the first time in 2009, is intended for advanced undergraduates and will be taught at the Morningside Campus.

Until I started this course, no instruction in virology had been offered at the Morningside Heights campus of Columbia University since the late 1980s. This is a serious omission for a first-class University. Sending graduates into the world without even a fundamental understanding of viruses and viral disease is inexcusable.

Course enrollment has steadily increased: 45 students in the 2009, 66 students in 2010, and an amazing 88 students this year. I am gratified that so many students want to learn about the world of viruses. From the photo you can see that the classroom is full, so if interest in the course continues to increase, we will need a larger room.

Most readers of virology blog will not be able to sit in on each lecture – but you can still watch every one of them. You will find a videocast of each lecture at the course website, at my page on Vimeo, and at iTunes University. An archive of the 2011 version of this course is available online or at iTunes University. I will announce when each lecture is posted on Twitter and Google+. Virology is a rapidly moving field, so rest assured that this year’s version of the course will be different.

The goal of Virology W3310 is to provide an understanding of how viruses are built, how they replicate and evolve, how they cause disease, and how to prevent infection. After taking the course, some of the students might want to become virologists. The course will also provide the knowledge required to make informed decisions about health issues such as immunization against viral infections.

Thanks to the internet, the information in my virology course is accessible to everyone.

After we discussed newly discovered entry factors for ebolavirus and hepatitis C virus on TWiV 166, the New York Times covered part of the story in Key protein may give Ebola virus its opening. Given my recent interest in the case fatality ratio of avian influenza H5N1, I was intrigued by the following introductory statement:

Of the pathogens that keep worried scientists awake at night, few rival Ebola for ruthless efficiency. The virus contains just seven genes, yet it manages to kill up to 90 percent of the people it infects.

Is it true that the fatality rate of ebolavirus is ‘up to 90 percent’? According to the WHO page on Ebola haemorrhagic fever,

Zaïre, Sudan and Bundibugyo species have been associated with large Ebola haemorrhagic fever (EHF) outbreaks in Africa with high case fatality ratio (25–90%) while Côte d’Ivoire and Reston have not. Reston species can infect humans but no serious illness or death in humans have been reported to date.

There have been roughly 1850 recorded cases with over 1200 deaths since ebolavirus was discovered, an average fatality rate of 65%. But have there been only 1850 human infections?

The answer is clearly no. The results of several serological surveys have shown that many individuals have antibodies against Zaire ebolavirus – purportedly the most lethal. The results of one study revealed antibodies in 10% of individuals in non epidemic regions of Africa. A similar seroprevalence rate (9.5%) was reported in villages near Kikwit, DRC where an outbreak occurred in 1995. In addition, a 13.2% seroprevalence was detected in the Aka Pygmy population of Central African Republic. No Ebola hemorrhagic fever cases were reported in these areas.

A more recent study examined sera from 4,349 individuals in 220 villages in Gabon. Antibodies against Zaire ebolavirus were detected in 15.3% of those tested, with the highest levels in forested regions (see map). The authors believe that the seropositive individuals had mild or asymptomatic ebolavirus infection:

The high frequency of ‘immune’ individuals with no disease or outbreak history raises questions as to the real pathogenicity of ZEBOV for humans in ‘natural’ conditions.

These findings indicate that the fatality rates of Zaire ebolavirus that are quoted widely are likely to be vast overestimates. Why the infection is more lethal during outbreak conditions is not known. One possibility is related to the size of the viral inoculum received. During outbreaks the virus is spread by contact with the blood, secretions, organs or other body fluids of infected individuals, which contain very large quantities of virus. In contrast, infections in nature – by contact with contaminated fruit, for example – may involve far less virus.

Whether we are discussing avian H5N1 influenza, ebolavirus, or even the fictitious MEV-1, do not assume that widely quoted fatality rates are correct – check the scientific literature!

Related:

Should we fear avian H5N1 influenza?

Becquart P, Wauquier N, Mahlakõiv T, Nkoghe D, Padilla C, Souris M, Ollomo B, Gonzalez JP, De Lamballerie X, Kazanji M, & Leroy EM (2010). High prevalence of both humoral and cellular immunity to Zaire ebolavirus among rural populations in Gabon. PloS one, 5 (2) PMID: 20161740

The finding of viral nucleic acid sequences in illegally imported wildlife products has attracted the attention of the New York Times, which published an article entitled From the jungle to J.F.K., viruses cross borders in monkey meat. It begins with a scary scenario:

This may read like a passage from a Richard Preston novel, but all an enterprising virus needs to do is jump aboard a traveling human or animal, and bam — the potential for a catastrophe akin to the 1918 Spanish influenza pandemic could emerge. That’s why the Centers for Disease Control and the Wildlife Conservation Society decided to undertake their first surveillance efforts for diseases of animal origins crossing in the United States.

However, the PLoS One paper that is cited in support of this story found short viral sequences, not viruses, in material confiscated at several airports. The authors of this report extracted nucleic acids from confiscated specimens (which included fresh, raw, lightly smoked or dried samples from chimpanzees, mangabeys, guenons, and rats) and screened them for pathogen nucleic acids by polymerase chain reaction (PCR). The authors note that “RNA quality was low with a predominance of degraded, low molecular weight fragments in the samples”. They identified small PCR DNA products from the genomes of simian foamy virus (a retrovirus) and herpesviruses in 15 out of 48 specimens. No complete viral genomes or infectious viruses are reported in the paper.

Perhaps the Times was mislead by the title of the PLoS One paper: Zoonotic Viruses Associated with Illegally Imported Wildlife Products. The abstract is similarly misleading:

Pathogen screening identified retroviruses (simian foamy virus) and/or herpesviruses (cytomegalovirus and lymphocryptovirus) in the NHP samples.

The authors correctly report their results at the end of the introduction:

Here we report finding sequences of simian retroviruses and herpesviruses in bushmeat confiscated at five US airports

Infectious viruses might accompany imported smoked monkey heads, dried bats and rat parts. The presence of small fragments of viral nucleic acids suggests that these materials were indeed once infected. But it is a large leap of faith to suggest that infectious agents are still present. It’s a good idea to use PCR to screen such material, but it is important to be specific when reporting the results. My suggestions for revised titles: Zoonotic viral sequences associated with illegally imported wildlife products (PLoS One) and From the jungle to J.F.K., small fragments of viral nucleic acids cross borders in monkey meat (NY Times). Far less interesting, but factually correct.