TWiV 396: Influenza viruses with Peter Palese


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TWiM 41: ICAAC live in San Francisco




TWiV 103: Shots with LJ Tan

Hosts: Vincent Racaniello, Alan Dove, and LJ Tan




Links for this episode:

Weekly Science Picks

Alan – BioGene, an iApp
Vincent – The Vertical Farm by Dickson Despommier


Universal influenza vaccines

The need to re-formulate the influenza virus vaccine in response to viral antigenic drift and shift makes for complex logistics of vaccine production and administration. Surveillance programs must be conducted each year to identify strains that are likely to predominate and cause disease. Wouldn’t it be simpler if a single vaccine could be developed that would confer protection against a broad range of viral strains? Results from the past year suggest that such a vaccine might be closer than previously thought.

The influenza viral HA protein consists of a globular head atop a stem that is embedded in the virion membrane (figure). Most protective antibodies are directed against the head of the HA molecule. Rare antibodies that block infection with a broad range of influenza virus strains are directed toward the conserved stalk of the viral surface glycoprotein HA. This observation was taken a step further by showing that sequential immunization with different viral HAs, or with HA lacking the globular head, induce broadly neutralizing antibodies. Peter Palese discussed these approaches on TWiV #102.

In another approach, neutralizing antibodies have been induced by immunizing first with plasmid DNA, followed by a boost with recombinant adenovirus encoding the HA protein. Mice were immunized first with plasmid DNA encoding an H1 HA from the 2006-2007 influenza season, then boosted with a recombinant adenovirus encoding the same HA protein. Sera from immunized mice neutralized strains of H1N1 influenza virus dating to 1934, as well as H2N2 and H5N1 viruses. When inoculated with a 1934 H1N1 virus, immunized mice were protected from lethal disease. Immunization of ferrets with a similar regimen also protected these animals from lethal disease. Broadly neutralizing antibodies were elicited in nonhuman primates by this prime-boost regimen.

Both the plasmid DNA and the recombinant adenovirus encoded the full-length HA protein, with both the globular head and fibrous stem. However, the broadly neutralizing and protective antibodies were directed against the stem. Anti-HA stem antibodies were also identified in monkeys that had been immunized with the prime-boost combination.

Why doesn’t the seasonal influenza vaccine elicit broadly neutralizing antibodies? These vaccines induce antibodies that almost exclusively bind the variable head of the HA, not the conserved stem. The reason probably lies in how the vaccines are prepared: virions are inactivated by treatment with detergent and formaldehyde, a process that destroys the particle. Consequently, the vaccine contains mainly HA and NA and not other components that can help shape a more diverse antibody repertoire. In contrast, it is known that plasmid-based priming can stimulate B cells to produce a more diverse set of antibodies.

The strategy of priming with plasmid DNA followed by boosting with recombinant adenovirus will likely be evaluated in clinical trials for the ability to protect against natural infection with influenza virus. The possibility of a broadly protective influenza virus vaccine that would be taken perhaps every 10-20 years is rapidly becoming a reality.

Wang TT, Tan GS, Hai R, Pica N, Petersen E, Moran TM, & Palese P (2010). Broadly protective monoclonal antibodies against H3 influenza viruses following sequential immunization with different hemagglutinins. PLoS pathogens, 6 (2) PMID: 20195520

Wei CJ, Boyington JC, McTamney PM, Kong WP, Pearce MB, Xu L, Andersen H, Rao S, Tumpey TM, Yang ZY, & Nabel GJ (2010). Induction of broadly neutralizing H1N1 influenza antibodies by vaccination. Science (New York, N.Y.), 329 (5995), 1060-4 PMID: 20647428

TWiV 102: Catch me if you can in Munich





Links for this episode:

Weekly Science Picks

Katharina – Deutsches Museum
Vincent – HHMI holiday lectures on science (thanks Judi!)


Swine influenza A/Mexico/2009 (H1N1) update

phases5-6Here is an update on the global swine flu situation as of 29 April 2009.

Not surprisingly, laboratory confirmed case counts continue to rise globally. There are 91 cases in the US in 10 states (Arizona, California, Indiana, Kansas, Massachusetts, Michigan, Nevada, New York, Ohio, Texas). There has been a laboratory confirmed fatal case in Texas, in a Mexican toddler visiting the US with his family. Eight other countries report a total of 57 laboratory confirmed cases, with no changes in the numbers in Mexico. Other countries reporting cases are Austria (1), Canada (13), Germany (3), Israel (2), New Zealand (3), Spain (4) and the United Kingdom (5). Globally nine countries have confirmed 148 cases with 2 deaths.

These numbers might not appear to justify the enormous reactions of health agencies such as CDC and WHO. Remember that there are probably many more infections with this virus, not only in the countries that report isolation of the virus but in other areas as well. It will likely take several weeks before we have a good appreciation for how extensively the virus has spread.

On Tuesday WHO raised the level of influenza pandemic alert from phase 4 to phase 5. According to WHO, “Phase 5 is characterized by human-to-human spread of the virus into at least two countries in one WHO region. While most countries will not be affected at this stage, the declaration of Phase 5 is a strong signal that a pandemic is imminent and that the time to finalize the organization, communication, and implementation of the planned mitigation measures is short.” WHO regions include African, European, Eastern Mediterranean, Americas, Southeast Asia, and Western Pacific. The cases in 4 European countries fulfilled the requirement for moving to phase 5.

Incredibly, Egypt began slaughtering 300,000 pigs as a ‘precautionary measure’ against the spread of swine flu. Farmers will not be compensated because the meat will be sold for consumption. This is a somewhat controversial move, because it is highly unlikely that pigs anywhere will play a role in further spread of the virus, which is now adapted to humans.

Sequences of viral RNAs from 20 swine flu isolates have now been posted on the NCBI website. Included are isolates from California, Texas, New York, Ohio, Kansas, and Germany (taken from a tourist who returned from Mexico). It is difficult to understand why RNA sequences of none of the Mexican isolates have  been posted, which would enable us to determine if the viruses in that country are different from the others. However, examination of the sequences of the New York and German isolates, which presumably originated in Mexico, reveal no significant differences with sequences from other isolates. From this information I conclude that the apparent higher virulence of swine flu in Mexico is not a consequence of a genetically diverged virus.

Other interesting information that can be gleaned from sequence information is contained in a statement from CDC: “…the HA, PB2, PB1, PA, NP, NS genes
contain gene segments from influenza viruses isolated from swine in North America [such as, A/swine/Indiana/P12439/00], while the NA and M genes are most closely related to corresponding genes from influenza viruses isolated in swine population in Eurasia.
However, the NA and M genes from 2 swine virus isolates from America are also closely related to the novel H1N1 virus (A/swine/Virginia/670/1987, A/swine/Virginia/67a/1987), if a reasonable nucleotide substitution rate is accepted. Thus, H1N1 from Mexico may be a swine flu virus strain of entirely American origin, possibly even of relatively ancient origin.” In the coming days I will attempt to construct a history of the evolution of swine influenza. In the meantime it may well be that this new human strain emerged from the US, as did the 1918-19 pandemic virus.

It is curious that CDC originally asserted that the new swine influenza virus inherited genes from human, pig, and bird viruses. Dr Anne Schuchat made this statement during a press conference on 23 Apr 2009, noting that  “Preliminary testing of viruses from the 1st 2 patients shows that they are very similar. We know so far that the viruses contain genetic pieces from 4 different virus sources. This is unusual. The 1st is our North American swine influenza viruses. North American avian influenza viruses, human influenza viruses, and swine influenza viruses found in Asia and Europe. That particular genetic combination of swine influenza virus segments has not been recognized before in the US or elsewhere.”

I am not sure why the sequence information now available indicates a very different origin for these viruses.

Although many still describe this virus as swine flu, it is technically no longer a pig virus – having acquired the ability to be transmitted among humans and cause disease, it is now a human virus. I realize that the official strain names are cumbersome (A/Mexico/4482/2009 [H1N1]), and therefore it is likely that we will be using ‘swine flu H1N1’ at least until the next pandemic.

Swine flu: Questions and answers


Q: Can you please tell me why there is a flu “season”.

Influenza is seasonal in temperate climates because conditions found during winter, low temperature and humidity, favor spread of the infection. In the dry conditions of winter, virus-laden respiratory droplets remain small and travel further. When humidity is high, these droplets take on water and hence do not move well through the air, limiting transmission. However, influenza can be isolated all year round from most large cities. Furthermore, influenza occurs throughout the year in tropical climates. Finally, influenza in swine is not seasonal. These observations indicate that other modes of influenza transmission – via direct contact or contaminated objects – may play important roles under some conditions.

Q: So far, people aren’t even dying in New York. What about Dengue Fever, spread by mosquito like Malaria, but with only a 1% fatality rate? With CDC estimates at about 100,000,000 cases a year, that’s 100,000 dying people worldwide. (It might be higher, because the fatality rate in developing countries is about 5%). In Puerto Rico, about 2-3 people a week die from Dengue….each year 350-500 million cases of malaria occur worldwide, and over one million people die, most of them young children in sub-Saharan Africa. In areas of Africa with high malaria transmission, an estimated 990,000 people died of malaria in 1995 – over 2700 deaths per day, or 2 deaths per minute. Apparently, unless people are dying in New York, a disease is not our problem….again from the Centers for Disease Control and Prevention, the number of alcohol-induced deaths in the U.S., excluding accidents and homicides, in 2005 was 21,634. Here’s another statistic, the number of Lyme Disease infections in the U.S. for 2005 were 23,305.

Now I realize these questions are short sighted and have made my own responses based on my limited knowledge but I would like to hear an answer from someone like yourself who is more knowledgeable on the subject.

A: Influenza is a serious disease, and if the current swine flu virus sparks a pandemic, it could lead to millions of deaths. Influenza is a preventable disease – an effective vaccine and antiviral drugs are available. This is why the outbreak is receiving much attention, so health officials can decide whether to produce a vaccine against this new strain. Some of the infectious diseases that you mention are not at the moment preventable, but they might be one day. Vaccines against dengue and malaria are under development – they have not been as straightforward to devise as an influenza vaccine. And a vaccine against Lyme disease was licensed but recently removed from the US market.

Q: Should we actively seek exposure to this wave of a relatively mild flu in hopes that some immunity protection will be afforded to a possible more deadly mutation in the fall?

A: Absolutely not. First, it is not certain that infection with the past season’s human H1N1 strain would confer any protection – I have not yet see data to answer the question one way or the other. Second, the infection might not result in mild disease – for any number of reasons it could be more severe than you think.

Q: Can we be absolutely metaphysically certain this is not a genetically engineered virus/viruses? Dr. Marie Gramer at the University of Minnesota, who has actually examined this virus first hand, and has probably the largest swine flu virus library in the US at the University of Minnesota, states that this virus isn’t even close to being serially homologous with any others in her library. It is a radically different entity. There has never been a virus with two swine flu, one avian and one human component. Are there sufficient grounds, in light of all this, to be absolutely positive these have not been genetically engineered?

A: The A/California/09/2009 (H1N1) strain, and related viruses from other parts of the world, were not genetically engineered. Sequences of the viral RNAs reveal that they are very similar to viruses currently circulating in pigs. The particular combination of RNA segments has not been previously observed, but the sequence of each viral RNA is recognizable as derived from swine. More importantly – no one on earth would know how to engineer a swine influenza virus that could be transmitted among humans and cause respiratory disease and death. Only evolution can select viruses with this property.

Q: There are reports of large corporate hog farms in the district where the first case of the flu originated. Anything between 50,000 to 950,000 hogs are supposed to be reared and processed in these farms. Could there be a link? Would it statistically increase the possibility of reassortants emerging from these farms?

A: It is my understanding that pigs on commercial farms in Mexico are vaccinated; although it is not the same strain this might confer some protection. The private pig farms, however, do not use the vaccine, and could be the source of the virus. The larger the farm, the more swine viruses are present, and the more chance for contact between pigs and humans, leading to more likely virus transfer from pig to human.

Q: What, if any, is the structural comparison of the current H1N1 strain with a swine flu outbreak in 1998 in the US?

A: In 1998 an H3N2 influenza virus was introduced into pigs in the US. This virus is very different from the current swine flu strain (H1N1) which resembles more recently circulating H1N1 swine viruses.

Q: It is said that human, avian and swine flu viruses have got shuffled together to produce the current type. How does that happen – meaning how do these three get together? Why are pigs needed for this? Can it happen in other organisms too?

A: Pigs are good hosts for influenza viruses because their respiratory tract can be infected with both human and avian influenza viruses (as well as swine flu of course). One reason why pigs are so susceptible is that their cells bear receptors for both human and avian influenza viruses. When two influenza viruses infect a cell, the viral genes mix and the new viruses produced can have genes from both infecting viruses. Influenza viruses infect many other animals, including birds, horses, dogs, cats, whales, and seals. Aquatic birds are the natural reservoir of the virus; they harbor all known HA and NA subtypes and may also serve to produce new strains.

Q: Should this be considered a prime candidate for next winters flu season?

A: It depends on the extent of spread of the virus in the southern hemisphere. A decision to make a vaccine must be made very soon, so that the vaccine can be deployed in the fall. If the virus continues to spread in the next week I would presume that vaccine production will go forward. It probably already is.

Many readers are perplexed by the unusual severity of the outbreak in Mexico. The following readers wonder what non-viral conditions might impact on disease severity:

Q: You mentioned in a TWiV that a theory about the mortality in the 1918 influenza stemmed from a concurrent infection with a bacteria that weakened the bodies immune response. Is that a possibility here? I only ask because you mentioned people living in such close proximity–already present flora in the lungs+pollution+new viral infection? Or could there be another virus, in addition to H1N1, causing an additional immunological insult?

Q: How about the delay in access to quality health care or laxity on the part of patients to seek treatment as they ignore the symptoms of early disease just as another case of “cold”? Combine it with secondary bacterial infections and/or high pollutant levels and low immunity levels, hence high mortality!

Q: I have a third theory about why fatalities could be higher in Mexico City: that town has some of the worst air quality in the world. I’m just speculating here, but it seems at least plausible that high smog and particulate pollution levels could tip some patients over the edge, turning an unpleasant but otherwise survivable case of the flu into a deadly viral pneumonia.

Q: It is too early to fully understand the current situation, but I would nevertheless draw your attention to the possibility that the very important pollution level and the high altitude of Mexico City (2,250 meters) can be partly responsible for the severity of swine influenza cases reported in Mexico. Remember that to date, all cases reported elsewhere in the world seem rather benign, or at least non-fatal. If this hypothesis is true, the severity of a pandemic outbreak may well be less severe than that suggests by the seriousness of the cases observed in Mexico City. Indeed, polluted cities such as Mexico City or located more than 2,000 meters are not numerous. I obviously do not know the nature of complications that resulted in the death of Mexican patients, but this knowledge could lead to a better understanding of how pollution and altitude should be considered as aggravating factors in the evolution of infection. Obviously, it is possible that the global death toll increase, but if, in the coming days, the severity of cases seen outside Mexico remains similar to what it is today and if it is confirmed that it is really the same viral strain, pollution and altitude could be important explanations of the current differential severity observed.


A: These are all reasonable and possible scenarios, but there is no evidence yet to support any of them. I am waiting for the viral RNA sequence for the Mexican strains to determine if they differ in any way from other isolates. However, there are few differences between the New York strains and those from other parts of the US. If the NY virus came from Mexico, this would imply that the differences in the virus do not account for the lethality in Mexico. The small amount of sequence available from a German isolate, from a tourist who visited Mexico, also shows that it is nearly identical to the US strains.

There has now been a laboratory confirmed fatal case in Texas, in a Mexican toddler visiting the US with his family. This should be viewed as part of the Mexican statistics but since the child died in Texas it goes into the US case count.

A comment from Mystery Rays:

I still think the most likely reason for the apparent difference in Mexico and the US is the missing denominator, rather than any host or environmental factor. That is, I suspect that the virus is much more widespread in Mexico City than the authorities know. Because they almost entirely tested severely ill hospitalized patients, it’s not surprising that they found high mortality rates; if they were also testing mild cases (as is happening in the US and elsewhere) I think they would likely find a very widespread infection with a low mortality rate.

A: I agree that it is still too early in the epidemic, and the numbers needed to determine the percent fatality are not yet available. Remember that the fatality of seasonal influenza is about 0.1%; for the 1918-19 pandemic it was about 2.5%. Considering the laboratory confirmed cases in Mexico, there have been 7 deaths out of  26 cases which would be a fatality rate of 30% – anomalously high and probably incorrect for the reasons above.

Q: With respect to differences in mortality/pathogenesis between Mexico and the US, what about the extent of vaccination? Certainly there’s a big push in the US to get regular vaccinations. Those who have been vaccinated have had multiple exposures to other H1N1 viruses.

A: I am not aware of how immunization rates in Mexico compare to those in the US, nor of the extent of circulation of other human H1N1 strains. This could play a role if such immunization or exposure lead to protection against the new influenza strain. CDC asserts that “Vaccination with seasonal influenza vaccine containing human influenza A (H1N1) would not be expected to provide protection against swine influenza A (H1N1) viruses”, but I have not seen the data on possible cross-protection.

Q: There has been no mention, save a passing comment from the media on pneumonia, of the pathology associated with this influenza pandemic.  Could it be necrotizing bronchial interstitial pneumonia or is that only associated with H5N1?  What about MODS or Cyanosis? What, essentially, is killing our Mexican neighbors?

A: I have not seen any information on the type of pathology observed in the Mexican victims. To date interstitial fibrosis has only been a feature of infection with H5N1 viruses. If anyone has such information, please share it with us.

Q: Regarding your first theory of why the non-Mexican cases are milder: “…infection with the current circulating human H1N1 strain might confer some protection…” I’m just a layman, and so I’m wondering exactly how something like that would work.  What percentage of (say) American victims would likely have this benefit?  Enough to explain the discrepancy?  Why wouldn’t we expect a similar number of “protected” persons in Mexico? Also, regarding the “air pollution theory”, haven’t their been deaths inside Mexico, but outside Mexico City?  Perhaps in locales with comparatively pristine air?

A: Virus infection stimulates the production of host proteins called antibodies, which bind to the virus and prevent its from infecting cells. Each year, influenza virus strains change so as to avoid binding by such antibodies. However, some of the antibodies produced by infection with the previous year’s strain confer some protection against the new strain, and hence disease may be milder. A pandemic strain is completely different and not affected at all by existing antibodies in the population. Antibodies produced in response to infection with previous human H1N1 strains might recognize the swine influenza virus and block infection. Whether or not this is the case is not known.

It is my understanding that most laboratory confirmed deaths have been in Mexico City (if anyone has more detailed information, please send it to virology blog). But with just seven laboratory confirmed deaths from swine influenza in Mexico, it is far too soon to reach a conclusion.

Q: A New Zealand blogger posits that getting swine flu now could be beneficial if this becomes a pandemic. (“How Swine Flu Could Save Your Life” – ) Clearly, he makes some poor leaps of logic, but he presents what seems to be a reasonable point re: cow pox and small pox….

Could getting the swine flu now help to weather future, more virulent strains?

A: Possibly; see the discussion above.

Since this flu also has a bird flu heritage, could catching (and surviving) it help with a possible H5N1 outbreak in the future?

No. As far as can be determined from examining the sequences of the viral RNAs, it does not appear that the virus has any genes from an avian strain. Furthermore, the  HA is very important for protective immunity, and the hemagglutinin (HA) subtypes are completely different: H1 vs H5.

Using Google to track influenza




