TWiV 348: Chicken shift

On episode #348 of the science show This Week in Virology, Vincent and Rich discuss fruit fly viruses, one year without polio in Nigeria, and a permissive Marek’s disease viral vaccine that allows transmission of virulent viruses.

You can find TWiV #348 at

Permissive vaccines and viral virulence

chicken farmA permissive vaccine prevents disease in the immunized host, but does not block virus infection. Would a permissive vaccine lead to the emergence of more virulent viruses?

This hypothesis is based on the notion that viruses which kill their hosts too quickly are not efficiently transmitted, and are therefore removed by selection. However a vaccine that prevents disease, but not viral replication in the host, would allow virulent viruses to be maintained in the host population. It has been suggested that in this scenario, viruses with increased virulence would be selected if such a property aids transmission between hosts.

On the surface this hypothesis seems reasonable, but in my opinion it is flawed. One problem is that increased transmission might not always be associated with increased virulence. The more serious flaw lies in making anthropomorphic assessments of what we think viruses require, such as concluding that increased viral transmission is a desired trait. Our assumptions fail to recognize the main goal of evolution: survival. Evolution does not move a virus along a trajectory aimed at perfection. Change comes about by eliminating those viruses that are not well adapted for the current conditions, not by building a virus that will fare better tomorrow. All the viruses on Earth today transmit well enough, or they would not be here; yet some kill their hosts clearly much faster than others. The fact is that humans have little understanding of what drives virus evolution in large populations. Our assumptions of what constitute the selective forces are usually tainted by anthropomorphism.

This long preamble is an introduction to a series of findings which are purported to support the idea that permissive vaccines (the authors call them ‘leaky’ and ‘imperfect’ vaccines but I dislike both names because they imply defects) can lead to the selection of more virulent viruses. The subject of the paper is Marek’s disease virus (MDV), a herpesvirus that infects chickens. MDV is shed from feather follicles of infected chickens and is spread to other birds when then inhale contaminated dust. Vaccines have been used to prevent MDV infection since the early 1970s. These vaccines prevent disease, but do not block viral replication, and vaccinated, infected birds can shed wild type virus. The virulence of MDV has been increasing since the 1950s, initially from a paralytic disease, to paralysis and death. The authors wonder if the use of permissive Marek’s vaccines has lead to the selection of more virulent viruses.

To address their hypothesis, the authors inoculate vaccinated or unvaccinated chickens with a series of MDV isolates that range from low to high virulence. Unvaccinated chickens inoculated with the most virulent MDV died within a week and shed little virus. In contrast, most vaccinated birds survived infection with virulent viruses, and shed virus for the length of the experiment, 56 days.

A transmission experiment was done to determine if shed virus could infect other birds. The authors infected vaccinated or unvaccinated birds and asked if sentinel, unvaccinated chickens became infected. Unvaccinated birds died within 10 days after infection with virulent MDV, and did not transmit infection. In contrast, vaccinated birds survived at least 30 days, and co-housed sentinel animals became infected and died.

The experiments are well done and the conclusions are clear: more virulent Marek’s disease viruses replicate longer in vaccinated than unvaccinated chickens, and can be readily transmitted to other chickens. But these results do not prove that more virulent MDV arose because of permissive vaccines. Nor do the results prove in general that leaky vaccines lead to selection of more virulent viruses. The results simply show that a vaccine that does not prevent replication will allow transmission of virulent viruses.

To prove that vaccinated chickens can allow the selection of more virulent viruses, vaccinated chickens could be infected with an avirulent virus, and the shed virus collected and used to infect additional, vaccinated birds. This process could be repeated to determine if more virulent viruses arise. While the results of this gain-of-function experiment would be informative, they would be done in a controlled laboratory setting which would not duplicate all the selective forces present on a poultry farm.

The authors note that most human vaccines do prevent replication of infecting virus. They do not mention the one important exception: the Salk poliovirus vaccines. People who are immunized with the Salk vaccine can be infected with poliovirus, which will then replicate in the intestines, be shed in the feces, and transmitted to others. This behavior has been well documented in human populations, yet the virulence of poliovirus has not increased for the 60 years during which the Salk vaccine has been used.

I do not feel that these experimental results have general implications for the use of any animal vaccine. It is unfortunate that the work has been covered in many news sources with the incorrect implication that vaccines may be responsible for the emergence of more virulent viruses.

TWiV 284: By the pricking of my thumbs, something wicked this way comes

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

Why do viruses cause disease?

EvolutionVirulence, the capacity to cause disease, varies markedly among viruses. Some viruses cause lethal disease while others do not. For example, nearly all humans infected with rabies virus develop a disease of the central nervous system which ultimately leads to death. In contrast, most humans are infected with circoviruses with no apparent consequence. Is there a benefit for a virus to be virulent?

One explanation for viral virulence is that it facilitates transmission. However, a comparison of infections caused by two enteric viruses, poliovirus and norovirus, does not support this general view. Both viruses infect the gastrointestinal tract and are spread efficiently among humans by fecal contamination. However, norovirus infection causes vomiting and diarrhea, while poliovirus infection of the intestine is without symptoms (the rare invasion of the nervous system, and subsequent paralysis, is an accidental dead end). Both viruses have successfully colonized humans for many years, so why does only one of them cause gastrointestinal tract disease?

Two recent studies of bacterial virulence provide some clues about the evolution of virulence. In one a commensal strain of Escherichia coli was serially propagated in the presence of macrophages, which are cells of the immune system that take up and destroy the bacteria. After many such passages, bacterial clones were isolated that escape phagocytosis and killing by macrophages. These clones had also acquired increased pathogenicity in mice. In other words, the genetic changes that allowed the bacteria to evade the immune response also lead to increased virulence.

In another example of evolution to virulence, it was found the the bacterium Pseudomonas aeruginosa can sense the presence of competing gram-positive bacteria because the latter shed the cell wall component peptidoglycan. In response to this molecule, P. aeruginosa secretes proteins that kill the other bacteria. These secreted proteins also make the bacterium more virulent in a host – in their absence, the bacteria are less virulent. In other words, P. aeruginosa damages its host in an attempt to remove nearby bacterial competitors.

In both bacterial examples, virulence can be viewed as collateral damage: the consequence of evading the immune response, or killing off competitors. Being virulent was not the primary goal. This explanation for bacterial virulence is straightforward and compelling: virulence is not directly selected for during evolution but comes along for the ride. Can it be applied to viruses?

All eukaryotic viruses must encode at least one protein that antagonizes host immune responses, otherwise they would be eliminated. These immune evasion proteins are certainly virulence factors: in general, when they are deleted or altered, the capacity of the virus to cause disease in a host is reduced. Like bacterial virulence, viral virulence might be collateral damage incurred by having to evade immune responses. This hypothesis is attractive but seems overly simplistic. If the ubiquitous and benign circoviruses did not evade host responses, then they would be eliminated from the human population.

The reasons why some viruses are virulent and others are not remain elusive. It is possible to reduce viral virulence by mutation, but this type of experiment does not reveal why viruses cause disease. The inverse experiment would be more informative: to select from a population of avirulent virus those that can cause disease. The results of such an experiment would help to identify the selection pressures that allow viruses to evolve to virulence.

TWiV 262: Wrong form, right professor

On episode #262 of the science show This Week in Virology, Vincent returns to the University of Wisconsin – Madison to speak with Ann Palmenberg about her career in virology.

You can find TWiV #262 at

TWiV 233: We’re surrounded

On episode #233 of the science show This Week in Virology, Vincent, Rich, Alan and Kathy review aerosol transmission studies of influenza H1N1 x H5N1 reassortants, H7N9 infections in China, and the MERS coronavirus.

You can find TWiV #233 at

TWiM 8: Live in NOLA

This Week in Microbiology #8Hosts: Vincent Racaniello, Michael Schmidt, Stan Maloy, Andreas Baümler, Nicole Dubilier, and Paul Rainey.

Vincent, Michael, and Stanley recorded episode #8 of the podcast This Week in Microbiology live at the 2011 ASM General Meeting in New Orleans, with guests Andreas Baümler, Nicole Dubilier, and Paul Rainey. They spoke about how pathogens benefit from disease, symbioses between chemosynthetic bacteria and marine invertebrates, and repetitive sequences in bacteria.

Click the arrow above to play, or right click to download TWiM #8 (60 MB, .mp3, 87 minutes).

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H1N1 pandemic is over

The World Health Organization has declared the end of the pandemic caused by H1N1 influenza virus. According to Director-General Margaret Chan,

The world is no longer in phase 6 of influenza pandemic alert. We are now moving into the post-pandemic period. The new H1N1 virus has largely run its course.

As we enter the post-pandemic period, this does not mean that the H1N1 virus has gone away. Based on experience with past pandemics, we expect the H1N1 virus to take on the behaviour of a seasonal influenza virus and continue to circulate for some years to come.

According to the Director-General, levels and patterns of H1N1 transmission are now different from those observed during the pandemic. Out-of-season outbreaks are no longer being reported, and their intensity is similar to that seen during seasonal epidemics. In addition, multiple influenza viruses are being isolated in many countries, a pattern typical of many recent seasonal epidemics.

I take particular interest in what the Director-General believes did not happen:

This time around, we have been aided by pure good luck. The virus did not mutate during the pandemic to a more lethal form. Widespread resistance to oseltamivir did not develop. The vaccine proved to be a good match with circulating viruses and showed an excellent safety profile.

I continue to wonder why the Director-General, and many others, feel that influenza virus must change to a more lethal form. Although the four previous influenza pandemics occurred in multiple waves of increasing lethality, there is no evidence that they are a consequence of viral mutation. For example, the only virus available from the 1918 pandemic was rescued from an Alaskan influenza victim who was buried in permafrost in November of that year, when higher mortality was already evident. This makes it impossible to correlate any genetic changes in the virus with increased virulence. Viruses are available from different stages of the pandemics of 1957 and 1968, which also occurred in waves of increasing lethality, but to my knowledge the virulence studies have not been done.

I believe that a major selective force for viral evolution is the need to maintain efficient transmission among hosts. This may be achieved by any number of phenotypic changes, such as increases in stability and virion production. Changes in lethality might also lead to more effective transmission – for example, by inducing more severe coughing, the virus could be better transmitted among humans. But there is no genetic evidence that such changes have occurred during influenza virus pandemics.

How has the idea that influenza virus mutates to greater lethality permeated our popular culture? I don’t know the answer, but John Barry’s The Great Influenza is a prime suspect.

TWiV #74: Influenza with Professor Adolfo Garcia-Sastre

Hosts: Vincent Racaniello and Adolfo Garcia-Sastre

Vincent speaks with Adolfo Garcia-Sastre talk about the origin, pathogenesis, and prevention of the 2009 pandemic influenza H1N1 virus.

This episode is sponsored by Data Robotics Inc. Use the promotion code VINCENT to receive $50 off a Drobo or $100 off a Drobo S.

Win a free Drobo S! Contest rules here.

Click the arrow above to play, or right-click to download TWiV #74 (34 MB .mp3, 47 minutes)

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

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The D225G change in 2009 H1N1 influenza virus

sialic-acid-2Last year a mutation in the HA gene of the 2009 H1N1 influenza virus was identified in isolates from patients with severe disease. At the time I concluded that the emergence of this change was not a concern. Recently the Norwegian Institute of Public Health reported that the mutation, which causes a change from the amino acid aspartic acid to glycine at position 225 of the viral HA protein (D225G), has been identified in 11 of 61 cases (18%) of severe or fatal influenza, but not in any of 205 mild cases. Have these observations changed my view of the importance of this mutation?

The cell receptor for influenza A virus strains is sialic acid. Human influenza A strains bind preferentially to sialic acids linked to galactose by an alpha(2,6) bond, while avian and equine strains prefer alpha(2,3) linked sialic acids (pictured). Alpha(2,6) linked sialic acids are dominant on epithelial cells in the human nasal mucosa, paranasal sinuses, pharynx, trachea, and bronchi. Alpha(2,3) linked sialic acids are found on nonciliated bronchiolar cells at the junction between the respiratory bronchiole and alveolus, and on type II cells lining the alveolar wall.

The 2009 swine-origin H1N1 influenza virus is known to bind both alpha(2,3) and alpha(2,6) linked sialic acids. This is consistent with the ability of the virus to cause lower respiratory tract disease. The D225G change might be expected to increase affinity for alpha(2,3) linked sialic acids. However, it is not known if increased binding affinity correlates with higher infectivity and pathogenicity. It’s equally likely that high affinity binding might restrict the movement of the virus in lung tissues by causing retention of the virus on nonsusceptible cells.

One view of the D225G mutation is that it is spreading globally and causing more severe disease. However there is no evidence in support of this hypothesis. According to WHO, viruses with the D225G change have been found in 20 countries since April 2009, but there has been no temporal or geographic clustering. As of January, the HA change has been identified in 52 sequences out of more than 2700. Furthermore, the authors of the Norwegian study write, “Our observations are consistent with an epidemiological pattern where the D225G substitution is absent or infrequent in circulating viruses, with the mutation arising sporadically in single cases where it may have contributed to severity of infection”.

One explanation for the sporadic emergence of influenza viruses with the D225G change is that they are selected for in the lower respiratory tract where alpha(2,3) sialic acids are more abundant than in the upper tract. Such selection might be facilitated in individuals with compromised lung function (e.g. asthmatics, smokers) or suboptimal immune responses, in whom the virus more readily reaches the lung. One way to address this hypothesis would be to compare the HA at amino acid 225 of viral isolates obtained early in infection, from the upper tract, with isolates obtained from the lower tract late in disease. However such paired isolates have not yet been obtained. But whether the presence of viruses with D225G increases viral virulence is unknown. Many H1N1 isolates from cases of fatal or severe disease do not contain this amino acid change.

There is an alternative explanation for the isolation of at least some influenza viruses with the D225G change: it is selected by propagation in embryonated chicken eggs. This selection occurs because cells of the allantoic cavity of chicken eggs have only alpha(2,3) linked sialic acids. A change in receptor specificity does not occur when viruses are propagated in MDCK (canine kidney) cells, which possess sialic acids with both alpha(2,3) and alpha(2,6) linkages. Consistent with this hypothesis, WHO reports (pdf) that the D225G substitution in 14 virus isolates occurred after growth in the laboratory.

Studies on the binding of influenza viruses to glycan arrays have shown that attachment is influenced not only by the linkage to the next sugar, but the type of sialic acid as well as the rest of the carbohydrate chain. The distribution of all the possible sialic acid containing sugars in the respiratory tract is unknown, as is the specific molecules that can support productive viral infection. The view that HA preferentially binds to either alpha(2,3) or alpha(2,6) linked sialic acids is likely to be overly simplistic: another casualty of reductionism.

Kilander A, Rykkvin R, Dudman SG, & Hungnes O (2010). Observed association between the HA1 mutation D222G in the 2009 pandemic influenza A(H1N1) virus and severe clinical outcome, Norway 2009-2010. Euro surveillance : bulletin europeen sur les maladies transmissibles = European communicable disease bulletin, 15 (9) PMID: 20214869

Takemae N, Ruttanapumma R, Parchariyanon S, Yoneyama S, Hayashi T, Hiramatsu H, Sriwilaijaroen N, Uchida Y, Kondo S, Yagi H, Kato K, Suzuki Y, & Saito T (2010). Alterations in receptor-binding properties of swine influenza viruses of the H1 subtype after isolation in embryonated chicken eggs. The Journal of general virology, 91 (Pt 4), 938-48 PMID: 20007353

Garcia-Sastre, A. (2010). Influenza Virus Receptor Specificity. Disease and Transmission American Journal Of Pathology DOI: 10.2353/ajpath.2010.100066