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TWiV 55: Mice lie, monkeys exaggerate

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twiv-200Hosts: Vincent Racaniello, Dick Despommier, Alan Dove, Jason Rodriguez, and Rich Condit

In episode 55 of the podcast “This Week in Virology”, the largest TWiV panel ever assembled takes on XMRV and chronic fatigue syndrome, 2009 chemistry Nobel prizes for ribosome structure, finding new poxvirus vaccine candidates, a brouhaha over leaked Canadian data on influenza susceptibility, and transmission of H1N1 influenza to a pet ferret.

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Links for this episode:

Weekly Science Picks
 Dick Nikon photomicroscopy contest winners at SciAm (Dick’s article on vertical farming)
Alan Make:
Rich BBC’s Planet Earth (DVD at Amazon)
Jason The Collider, the Particle and a Theory About Fate
Vincent An Epidemic of Fear and Misinformants at Wired Magazine

Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv or leave voicemail at Skype: twivpodcast. You can also send articles that you would like us to discuss to delicious and tagging them with to:twivpodcast.

Pandemic H1N1 influenza virus outcompetes seasonal strains in ferrets

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ferret-h1n1-coinfectionWhen more than one influenza A virus subtype is circulating in humans, as has been the case since 1977, there are several possible outcomes. The viruses might co-circulate, one virus might out-compete another, or co-infection of cells with two viruses can lead to the production of genetically distinct viruses by the process of reassortment of viral RNAs. Experiments have been done in ferrets to determine how the 2009 pandemic H1N1 strain interacts with seasonal H3N2 and H1N1 viruses.

Ferrets were intranasally co-infected with an H1N1 pandemic strain [Ca/04] and either a seasonal H1N1 virus [BR/59] or a seasonal H3N2 virus [BR/10].  One uninfected ferret was placed in the same cage (to allow contact transmission) and a second in another cage separated by a wire mesh (to allow aerosol transmission). They determined whether the viruses replicated in the animals by detecting viral RNA in nasal washes taken 1 day after infection using polymerase chain reaction (PCR), or by hemagglutination-inhibition assays to measure serum antibodies.

The results are striking. Both viruses replicate well in co-infected ferrets – look at panel C of the image above. The figure is a photograph of the DNA products of the PCR, separated by gel electrophoresis. Each lane shows the DNA corresponding to the individual influenza viral RNA. Panels A and B show the amplified DNAs from the nasal washes of two animals who were infected by contact. In these animals, the pandemic CA/04 virus replicates well (right half) while the seasonal H1N1 strain [BR/59] does not (left half).

When the nasal washes from the respiratory droplet contact ferrets were used to infect a new set of ferrets, only the pandemic CA/04 virus was detected; there was no evidence of the seasonal BR/59 or BR/10 viruses.

The pattern of the DNAs are distinct for each virus. For example, the PB2 DNA of BR/59 migrates more slowly on the gel than the PB2 DNA of CA/04 (panel C). Given these differences in migration of the DNAs, it would be easy to determine if there were reassortants in the co-infected ferrets. None can be detected by this analysis.

If these results were directly applicable to humans, we would predict that the 2009 H1N1 pandemic strain is not likely to reassort with the seasonal strains; and that it will out-compete those strains, which will eventually disappear. But we are not ferrets, and we don’t know whether these findings apply to humans. Nevertheless, the authors are allowed to speculate:

Although we must be cautious interpreting studies in the ferret model, it is reasonable to speculate that this prototypical pandemic strain, Ca/04, has all the makings of a virus fully adapted to humans.

In this case ‘fully adapted’ means that the pandemic strain replicates better than the seasonal strains or any reassortants that might arise in co-infections.

What did the press learn from this work? Reuters concluded:

And while a new study in ferrets suggested the virus spreads more quickly and causes more severe disease than seasonal flu, the good news is that it does not appear likely to mutate into a “superbug” as some researchers had feared.

Virologists consider mutation and reassortment to be two distinct phenomena. The mutation rate of the virus is determined by error-prone RNA synthesis. The host applies the selection pressure that enriches for a particular phenotype. The results of these studies reveal nothing about the ability of the virus to mutate.

Perez, D., Sorrell, E., Angel, M., Ye, J., Hickman, D., Pena, L., Ramirez-Nieto, G,, Kimble, B., & Araya, Y. (2009). Fitness of Pandemic H1N1 and Seasonal influenza A viruses during Co-infection PLoS Currents RRN1011.2.

Is the 2009 H1N1 influenza virus more dangerous than we think?

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Mustela_putorius_furoThe results of experiments comparing the virulence in animals of the 2009 H1N1 influenza virus with seasonal strains have spawned the headline Study Suggests H1N1 Virus More Dangerous Than Suspected. In my view, the best experiment is now being done in humans: infection of millions with the pandemic virus. The results show that the virus is no more virulent than last season’s H1N1 strain.

In mice, ferrets, and non-human primates, the 2009 H1N1 swine-origin influenza virus (S-OIV) replicated more efficiently, and caused more severe lesions in the lungs than a seasonal H1N1 virus. These findings lead the lead author of the study to comment:

There is a misunderstanding about this virus. People think this pathogen may be similar to seasonal influenza. This study shows that is not the case. There is clear evidence the virus is different than seasonal influenza.

I’m puzzled by this statement. As far as I know, the 2009 H1N1 strain has so far likely infected millions of people, and most have concluded that the disease is no more severe than seasonal influenza. Are mice, ferrets, and non-human primates more reliable indicators of influenza virus virulence than humans?

I agree that the 2009 H1N1 influenza virus does seem to multiply more extensively in the respiratory tract than a seasonal H1N1 strain, as does the 1918 virus. But how many influenza virus strains have been studied in such animals? There are probably others that can replicate in the lower tract of experimental animals but are not very pathogenic in humans.

Two other research groups published the results of similar experiments in ferrets. They both found that the 2009 H1N1 virus replicated to higher titers, and more extensively in the lower respiratory tract, than seasonal H1N1 influenza virus. One of the groups concluded:

Our results indicated that the 2009 A(H1N1) influenza virus replicates efficiently in the upper and lower respiratory tract of ferrets, is associated with mild or moderate clinical signs and pathological changes and is transmitted efficiently between ferrets via aerosols or respiratory droplets. These results are in agreement with observations in humans, where generally mild disease but relatively efficient human-to human transmission has been observed

In other words, although the 2009 H1N1 replicated more efficiently, and in different parts of the lungs than seasonal H1N1 virus, these authors felt the observations were consistent with the low virulence of the virus humans.

It’s worth noting that when the authors of the Nature paper infected miniature pigs with the 2009 H1N1 influenza virus, the virus replicated without clinical symptoms. This result emphasizes the importance of remembering that animals are models for studying virus infections; they rarely duplicate the effects of a virus infection in humans. Furthermore, the virulence of a virus strain may vary dramatically depending on the dose and the route of infection, as well as on the species, age, gender, and susceptibility of the host. Virulence is a relative property. Consequently, when the degree of virulence of two very similar viruses are compared, the assays must be identical.

Do we really want to conclude that the new H1N1 strains is ‘more dangerous’ than we think based on tests in animal models which may or may not accurately reflect what occurs in humans? The ongoing infection of millions of humans with the new H1N1 virus seems a better source of data – and so far the results indicate that the new pandemic strain is no more dangerous than seasonal influenza.

Of course, it is possible that a more virulent version of the 2009 H1N1 virus could emerge in the coming months – what will happen as this virus evolves in humans is anyone’s guess – but who knows if it will retain the ability to multiply in the lower tract? The only thing that is certain is that it will be a different virus from the one that has been studied in mice, ferrets, and small pigs.

Y. Itoh1 et al. (2009). In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses Nature adv. online pub 13 July 2009.

Maines, T., Jayaraman, A., Belser, J., Wadford, D., Pappas, C., Zeng, H., Gustin, K., Pearce, M., Viswanathan, K., Shriver, Z., Raman, R., Cox, N., Sasisekharan, R., Katz, J., & Tumpey, T. (2009). Transmission and Pathogenesis of Swine-Origin 2009 A(H1N1) Influenza Viruses in Ferrets and Mice Science DOI: 10.1126/science.1177238

Munster, V., de Wit, E., van den Brand, J., Herfst, S., Schrauwen, E., Bestebroer, T., van de Vijver, D., Boucher, C., Koopmans, M., Rimmelzwaan, G., Kuiken, T., Osterhaus, A., & Fouchier, R. (2009). Pathogenesis and Transmission of Swine-Origin 2009 A(H1N1) Influenza Virus in Ferrets Science DOI: 10.1126/science.1177127

TWiV 40: Tamiflu in the water

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twiv-200Hosts: Vincent Racaniello, Dick Despommier, and Alan Dove

On episode #40 of the podcast “This Week in Virology”, Vincent, Dick, and Alan consider Reston ebolavirus in swine, historical perspective of H1N1 influenza virus emergence and circulation, Tamiflu-resistant H1N1, Tamiflu in Japanese river waters, transmission of H1N1 virus in ferrets, and pneumonia and respiratory failure from H1N1 in Mexico.

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Click the arrow above to play, or right-click to download TWiV #40 (49 MB .mp3, 70 minutes)

Subscribe to TWiV in iTunes, by the RSS feed, or by email

Links for this episode:
Reston ebolavirus in Philippine swine
Historical perspective on H1N1 virus
Salk’s 1947 article on flu vaccine failure
Persistent legacy of 1918 H1N1 virus
Tamiflu resistant H1N1 virus (AP article)
Tamiflu in Japanese river waters
H1N1 infection of ferrets (article one and two)
Pneumonia and respiratory failure from S-OIV in Mexico
DNA-based equine WNV vaccine (thanks Peter!)
Fundamentals of Molecular Virology by Nicholas Acheson

Weekly Science Picks
Alan Coming to Life by Christiane Nusslein-Volhard
Dick Monsters Inside Me from Discovery Channel
Vincent Microbeworld

Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv or leave voicemail at Skype: twivpodcast

Influenza virus transmission

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sneezeInfluenza virus may be transmitted among humans in three ways: (1) by direct contact with infected individuals; (2) by contact with contaminated objects (called fomites, such as toys, doorknobs); and (3) by inhalation of virus-laden aerosols. The contribution of each mode to overall transmission of influenza is not known. However, CDC recommendations to control influenza virus transmission in health care settings include measures that minimize spread by aerosol and fomite mechanisms.

Respiratory transmission depends upon the production of aerosols that contain virus particles. Speaking, singing, and normal breathing all produce aerosols, while coughing and sneezing lead to more forceful expulsion. While coughing may produce several hundred droplets, a good sneeze can generate up to 20,ooo. Aerosolized particles produced by these activities are of different sizes. The largest droplets fall to the ground within a few meters and will transmit an infection only to those in the immediate vicinity. Other droplets travel a distance determined by their size. Those droplets 1-4 microns in diameter are called ‘droplet nuclei’; these remain suspended in the air for very long periods and may not only travel long distances, but can reach the lower respiratory tract. Inhalation of droplets and droplet nuclei places virus in the upper respiratory tract, where it may initiate infection.

The importance of aerosol transmission is illustrated by an outbreak of influenza aboard a commercial airplane in the late 1970s. The plane, carrying 54 persons, was delayed on the ground for three hours, during which time the ventilation system was not functional. Most of the travelers remained on board. Within 72 hours, nearly 75% of the passengers developed influenza. The source of the infection was a single person on the airplane with influenza.

Nasal secretions, which contain virus particles, are responsible for transmission by direct contact or by contaminated objects. An infected person will frequently touch their nose or conjunctiva, placing virus on the hand. Intimate or non-intimate contact (e.g. shaking hands) will transfer the virus to another person, who will then infect themselves by touching their nose or eyes. When contaminated hands touch other objects, the virus is transferred to them. In one study, 23-59% of objects from homes and day care facilities were shown to harbor influenza viral RNA. Others have shown that infectious influenza virus may be persist on paper currency for several weeks.

Influenza transmission can be reduced by covering your nose and mouth when coughing or sneezing, and by washing hands often with soap and water or alcohol-based hand cleaners. Note that CDC does not recommend the use of face masks for reducing viral spread. It is important to recognize that, in human infections, maximum levels of virus shedding may occur about a day before the peak of symptoms.

Ethical considerations preclude controlled influenza virus transmission studies in humans, and therefore animal models must be used. Ferrets are susceptible to many strains of influenza virus, and develop symptoms similar to those in humans. However these animals are costly and difficult to house, precluding their use in most large scale transmission studies. The guinea pig has recently been described as an alternative animal model for studying influenza virus transmission. These animals are susceptible to different viral strains, and the virus replicates to high titers in the respiratory tract. The guinea pig was used to show that transmission of influenza virus occurs by aerosols and through contaminated environmental surfaces. The efficiency of aerosol transmission in the guinea pig model is regulated by temperature and humidity.

Fabian, P., McDevitt, J., DeHaan, W., Fung, R., Cowling, B., Chan, K., Leung, G., & Milton, D. (2008). Influenza Virus in Human Exhaled Breath: An Observational Study PLoS ONE, 3 (7) DOI: 10.1371/journal.pone.0002691

Carrat, F., Vergu, E., Ferguson, N., Lemaitre, M., Cauchemez, S., Leach, S., & Valleron, A. (2008). Time Lines of Infection and Disease in Human Influenza: A Review of Volunteer Challenge Studies American Journal of Epidemiology, 167 (7), 775-785 DOI: 10.1093/aje/kwm375

Mubareka, S., Lowen, A., Steel, J., Coates, A., García‐Sastre, A., & Palese, P. (2009). Transmission of Influenza Virus via Aerosols and Fomites in the Guinea Pig Model The Journal of Infectious Diseases, 199 (6), 858-865 DOI: 10.1086/597073