Every couple of months a major flu paper appears purporting to reveal why the 1918 H1N1 virus was so horrifically virulent in comparison to the other pandemic viruses of the last century, H2N2 (1957 pandemic) and H3N2 (1968 pandemic). It's not just the H1N1 subtype of the influenza A virus that made it so deadly. There's still lots of H1N1 around, even as I tap this keyboard, but it isn't as virulent as the 1918 variety. Why not? If we knew the answer we might be able to spot a genetic change in circulating viruses indicating a turn toward virulence or find a drug or vaccine solution to treating its worst effects. And we would understand more about flu, a major public health virus. This week we were treated to another paper of this variety. Typical headline: Researchers unlock secrets of 1918 flu pandemic. Two questions immediately come to mind: have they found "the" secret? and if so, what is it? In my read of the paper I'd answer the first by saying we know more about what might be involved than we did before. It's excellent work. But it's obvious there are a lot of unknowns still and probably other ways this versatile virus can create havoc. And to what they found, that's a longer answer, so pull up a chair.
In 1918 we didn't know that influenza was caused by a virus. But we still have the virus from 1918 because of some ingenious (and some feel risky) work by scientists who fished out fragments of the virus's genetic material from some flu victims buried in permafrost (thus preserving the tissue) and reconstructing it. Once the genetic sequence was known it was possible to make the virus and manipulate it. That was done a few years ago and since then flu scientists have been trying to figure out what made it so deadly. The first suspicious fell on the hemagglutinin (HA) protein on the virus's surface. This is the part that "docks" to human or bird or some other host's cells and uses this as an entry way into the cell (for a more detailed description of the science see our previous posts here, here, here and the links in these posts). A virus's only goal is to make copies of itself and it does this by hijacking the genetic replication and protein making machinery of the host cell and therefore it needs access to the interior of the cell where this machinery is located. At first it appeared that it was some feature of the HA protein (there are 16 different subtypes of HA, numbered H1 to H16; these are the H-part of the subtype designations) that made it so deadly. When the 1918 H1 version was present the virus was highly virulent in mice. With a contemporary or laboratory version of an H1 (the kind that circulates today) the mice didn't get so ill. This was the first spate of papers proclaiming that "the secret" of the 1918 virus had been found. But the 1918 H1 didn't seem to have features associated with virulence in bird viruses, in particular, the extra basic amino acids at the cleavage site, something that practically defines the highly pathogenic bird flu virus, H5N1. So people kept looking for other factors that might be associated with pathogenicity. Recently it was show that the PB1 protein, part of the hijacking machinery the virus uses, was important for the 1918' virus's efficient replication in mouse lung and human airway cells. And now the newest paper to reveal "the secret." Newspaper articles are describing it as a combination of three 1918 flu genes, but as I read the paper it is four genes, all internal to the virus (that is, not on its surface, so they aren't "seen" by the part of the immune system that makes antibodies). The four proteins are the three that are part of the viral polymerase complex (PA, PB1 and PB2) and the nucleoprotein, NP.
Let's back up a minute. Influenza virus carries its own genetic blueprint in the form of RNA (our genetic blueprint and that of all other animals and many viruses is in DNA). Influenza is a negative-sense (or negative polarity) single-strandeded RNA virus. This means that the genetic material is present in a single strand or sequence of RNA (which is like DNA but one of its four letters is different). We have our genetic information in two matched strands that are genetic mirror images. Thus our genetic information is really present twice, once on each strand. We use the double strand to do two things. Make a copy of itself when the cell divides. And use one of the strands to direct the cell's protein making machinery to make the protein it codes for. The influenza virus needs to do the same thing: Make a copy of itself and make viral proteins. It does this from the single strand of RNA. Flu virus needs to make an intermediate mirror image single strand of RNA to make a copy of itself and to make protein (that's what makes it "negative sense"). The virus uses the host cell's protein making machinery but it has some important helper proteins of its own to hijack the host cell and carry out some of the functions it needs to make a copy of itself. The helper proteins are the four the new paper found were made the virus especially virulent when they came from the 1918 virus: PA, PB1, PB2 and NP.
Here are some more details. Viral RNA is rarely found "naked." It is usually dressed by being tightly intertwined with a protein, nucleoprotein. When viral genetic material directs the cell to make protein it needs or to make a copy of itself it does so in full dress uniform, i.e., as a ribonucleoprotein [RNA-protein complex], RNP. The other three proteins are also present in a combination, the RNA-dependent polymerase complex composed of three parts, PA, PB1 and PB2. Think of this as a machine that makes new viral RNA/RNP and new proteins like the hemagglutinin, more NP and PA, PB1, PB2 and a few other proteins the virus needs. It's called RNA-dependent to distinguish itself from most host polymerases which use DNA as their directing template.
The paper just published in the Proceedings of the National Academy of Sciences (Tokiko Watanabe, Shinji Watanabe, Kyoko Shinya, Jin Hyun Kim, Masato Hatta, and Yoshihiro Kawaoka, Viral RNA polymerase complex promotes optimal growth of 1918 virus in the lower respiratory tract of ferrets PNAS 2008 : 0806959106v1-pnas.0806959106) from the highly expert Kawaoka lab at the University of Wisconsin and the Universities of Kobe and Tokyo used two viruses, a 1918 H1N1 and a recent H1N1 (A/Kawasaki/173/2001; K173) that causes today's run-of-the-mill seasonal flu (which is still a nasty disease but not remotely as virulent as the 1918 variety). Using a technique called reverse genetics Kawaoka was able to mix and match genes from the 1918 and K173 viruses, substituting 1918 genes one at a time against the background of K173 genes. Here's a summary from the abstract of their paper:
We then assessed their virulence properties in ferrets, a model closely resembling humans in terms of sensitivity to influenza virus infection and pattern of spread after intranasal inoculation. Substitution of single genes from the 1918 virus in the genetic background of K173 virus did not markedly alter the pattern of infection. That is, the reassortants grew well in nasal turbinates, but only sporadically (if at all) in the trachea and lungs. One exception was the 1918PB1/ K173 reassortant, which replicated efficiently in lung tissues as well as the upper respiratory tract. A reassortant virus expressing the 1918 viral RNA polymerase complex (PA, PB1, and PB2) and nucleoprotein showed virulence properties in the upper and lower respiratory tracts of ferrets that closely resembled those of wildtype 1918 virus. Our findings strongly implicate the viral RNA polymerase complex as a major determinant of the pathogenicity of the 1918 pandemic virus. This new insight may aid in identifying virulence factors in future pandemic viruses that could be targeted with antiviral compounds.
In other words, the single substitution of PB1 allowed the K173 virus to grow in ferret lung and upper respiratory tract. No other single gene substitutions were able to do that. Substituting 1918 genes generally made the K173 virus less fit. This included the HA and NA genes, formerly thought to be where the virulence difference resided. For single gene differences, the only equally fit substitution was PB1. The important result, however, was that when the genes for the entire 1918 polymerase complex (PB2 and PA along with PB1) were substituted in K173 along with NP, the virus became 1918-class nasty for ferrets. These four genes converted the seasonal H1N1 into a 1918 H1N1. Of course the flu virus has only 8 segments and this replaced four of them, so one can say the "new" reassortant was already half 1918 virus. The more interesting biological fact is that these findings direct attention to the replication and protein translation part of the virus. It's not just any four proteins but four proteins that are functionally very closely related. This is a significant "clue." But it isn't "the" secret. We still don't know what it is about this combination that makes it so virulent nor can we be sure there aren't several other roads to virulence involving other proteins and combinations.
We're learning important things. This evidence strongly suggests that some feature of the polymerase complex (perhaps optimized for a particular RNP) allowed the virus to invade the lower respiratory tract and lung tissue of ferrets. Understanding that is a big step forward, a direction different than we have been looking up to now, where much work has gone into the difference between bird and human receptors in the upper and lower respiratory tracts. Whether the virulence of H5N1 bird flu follows this pattern or another we don't yet know. I presume a similar experiment is already underway. But there are still many other things we don't know, including the exact nature of the virulence. Knowing what is different about a virulent virus is only part of the story. We need to know why and how the differences are important. The virulence of 1918 virus or H5N1 is a biological property. It is not currently decipherable from genetic sequences. That is one important reason that providing genetic sequences but withholding viral isolates is a serious problem for flu scientists. We need the actual virus not just a string of letters. We can't solve this problem by sitting in front of a computer sieving published sequences. Not yet. Maybe not ever.
Perhaps the 1918 virus has given up one of its secrets. But it hasn't given up "the" secret. Not yet. Maybe not ever.
Three questions: First, regarding this statement -
" when the genes for the entire 1918 polymerase complex (PB2 and PA along with PB1) were substituted in K173 along with NP, the virus became 1918-class nasty for ferrets."
Question: How do today's less virulent H1N1 genes for this polymerase complex and NP differ from the original genes, and do the differences appear to be due to simple point mutations or some kind of reassortment or recombination mechanism?
My second question is related to this statement -
"This evidence strongly suggests that some feature of the polymerase complex (perhaps optimized for a particular RNP) allowed the virus to invade the lower respiratory tract and lung tissue of ferrets."
My Question: What do you mean 'invade'? My understanding of the function of the polymerase complex and NP is that it goes to work AFTER the virus invades the cell and takes over, that it is not part of the virus's machinery for attachment and insertion into the host cell, but part of its replicative machinery.
This would indicate that infection isn't the key, it's the efficiency with which the virus is able to replicate once inside the host cell.
Earlier posts on this site discussed at length the fact that the current avian influenza - H5N1 - has attachment proteins that bind primarily with one type of receptor found in the upper respiratory tract of birds. These receptor sites are found infrequently in the upper respiratory tract of humans, but are more prevalent in the lower respiratory tract of humans. (I hope I am remembering this right!!)
It was proposed that to become an efficiently transmittable human pandemic virus the attachment site would have to mutate into a form that attached to the slightly different receptors found commonly in the upper respiratory tract of humans.
However this recent study indicates that once the virus does attach to the receptor in the lower respiratory tract, it is so virulent that it easily spreads throughout the lungs causing the kind of pneumonia we have seen in victims.
My third question is if, because the lungs as an entry point are connected directly with the circulatory system, and the viruses then are capable of spreading quickly throughout the body infecting all other organs, then wouldn't they also be able to infect via this secondary route the mucous membranes of the upper respiratory tract and be able to be more easily spread via airborne droplets from coughing? My point is, do you still think the virus needs to mutate its attachment site to become a pandemic, and also if it does change that attachment site - but the true cause of its virulence is the polymerase complex and NP - is there any reason to believe that it will lose virulence once it does go pandemic?
Question: How do today's less virulent H1N1 genes for this polymerase complex and NP differ from the original genes, and do the differences appear to be due to simple point mutations or some kind of reassortment or recombination mechanism?
There are 25 to 35 amino acid differences in each of the genes. How they make a difference we don't know. It is likely that differences in one gene require some kind of parallel differences in the other two or three, that is, that the machine has to be fited together and optmized in some way, in what way, we don't know.
2nd question: Invade here means able to take up shop and replicate. We have talked quite a bit about the receptors here because that was the prevailing theory about why the virus didn't infect the lower tract very frequently. But we have been quite consistent that we didn't think there was enough data to establish this as we knew very little about the distribution of receptors in the various tissues. It was a good story but didn't have all the chapters to make it whole. Receptors clearly play a role but the relative importance isn't clear. "Infect" can mean a lot of things, but in general it means getting into a host cell and replicating efficiently there and then being able to infect other cells. Without the last part it is a dead end.
Third question: We don't know all the modes of transmission.. We've written a lot of posts about this. In particular we don't know if the g.i. tract might not be a mode (we know it is in some cases). The fact that it is certainly airborne as one route means it is very dangerous as this is hard to control. I have my doubts about fmoites as an important route but that's still open. Gastrointestinal is the last possibility and I see no reason it wouldn't be effective. So I'm not sure exactly what you are asking. Regarding the receptor part, I have raised questions about it on numerous occasions as we don't really know enough about this part of the equation. There has to be some way for the virus to get into the cell and the only question is whether it already has that (because that isn't really where the barrier is) or whtether it needs that and other things.
Finally, I never had the chance to reply to you about why I thought Tom and The Doctor were advancing a version of Lamackianism. You understand the role of natural variation correctly and my remarks were not directed at you. The other two commenters don't seem to, because they consistently talk as if the mutations didn't exist before the antiviral use. The places wheere there is the highest prevalence of resistance (e.g., Norway) are not under selective pressure. I don't have the previous comments here to quote you exactly the incriminating phrases, but if you do, go back and read them in that light.
Well reported. I think the benefits of testing like this outweigh the dangers of doing this kind of research. Let's just hope they have airtight controls.
We need to keep pandemic preparedness at the forefront of every business manager's mind. It won't go away so better start preparing.
but the gastrointestinal tract is not involved in normal
flu, only maybe H5N1 , right ?
so a 1918-virus may still emerge from today's
viruses (or in a military lab), but hopefully
won't be so virulent
since we have exposure to seasonal H1N1.
Thanks to the assumed Chinese Lab-escape in 1977 !
anon: Very little info or research on the g.i. route. Presumably it is not important for H1 - H3 subtypes. Are there other syndromes from g.i. flu virus infection? Probably not, but I doubt anyone has looked very hard. Regarding cross reactivity to H1N1, we have H1N1 outbreaks now. If virulence is not tied to the HA, then a virus with the 1918 polymerase complex is plausibly dangerous. But I don't worry much about lab accidents causing pandemics compared to Nature's laboratory.
Hi Revere. Thank you for the answers to my questions above, and also for clarifying that Lamarckian thing. I have one comment, fwiw, about this part of your answer:
"There are 25 to 35 amino acid differences in each of the genes."
Although a substitution point mutation wouldn't cause this kind of change in one single replicative cycle, the addition or subtraction of a single nucleotide during the RNA replication stage, especially one near the front of the chain, could cause a frameshift mutation that might account for such a large number of amino acid differences in a single mutation, I believe. However for this to become the "common" H1N1 sequence, such a mutation must have been favorable for the "new" virus as compared to its more lethal antecedents. Since it is generally considered more favorable for a pathogen's own life cycle if it does not entirely kill off its hosts (aka "the prudent parasite")that would make sense.
I also have a question about what you say here:
"It is likely that differences in one gene require some kind of parallel differences in the other two or three, that is, that the machine has to be fited together and optmized in some way, in what way, we don't know"
That seems to indicate that there would be more than simple natural random mutation and variation at work here, which of course we know can't really happen. Whereas in a regular cell there are mechanisms - enzymes and such - at hand to repair or adjust for "mistakes" during DNA replication or RNA transcription, would an invading virus be able to utilize such cell machinery to create parallel differences in the other two or three members of the polymerase complex? And if there were not such selective changes made, if it all occured by random chance mutations, what are the odds that all three polymerases could mutate more or less at the same time, so that each had 25 or so new amino acids that all worked together functionally, (even taking into consideration the vast numbers of viral replications that take place in a single individual.) Could these changes take place slowly over many different replicative cycles before the "machine was fitted together and optimized" and yet in the interim still have a virus able to infect and replicate effectively?
Or is it possible that the change in H1N1 came about through recombination with another virus, possibly that unknown "precursor virus" that has been suggested as being responsible for the initial milder first wave of the flu pandemic?
mary: We can only speculate at this point. The number of amino acid differences is given in teh paper but no details. However I think a frameshift would be easily spotted. They have the genetic sequence, not just the protein sequence. Remember, too, we don't know the origin of the 1918 virus but it is now geneally thought to have jumped from birds where the polymerase complex may have worked differently.
Regarding the moderation of virulence, we've discussed this here several times. Here's one post specifically on that topic:
Consider also that HIV, rabies and smallpox did not moderate their virulence. It's just not necessary for this to happen, although it might. In the flu case, it does appear that there has been a moderation of virulence in the case of the 1918 H1N1 and subsequent viruses derived from it.
Host restriction of avian influenza viruses at the level of the ribonucleoproteins
9/16/08 Annual Reviews of Microbiology--[full text]--Naffakh N, Tomoiu A, Rameix-Welti MA, van der Werf S.
Unité de Génétique Moléculaire des Virus Respiratoires, URA CNRS 3015, Institut Pasteur, Paris, 75015 France; email: firstname.lastname@example.org.
Although transmission of avian influenza viruses to mammals, particularly humans, has been repeatedly documented, adaptation and sustained transmission in the new host is a rare event that in the case of humans may result in pandemics. Host restriction involves multiple genetic determinants. Among the known determinants of host range, key determinants have been identified on the genes coding for the nucleoprotein and polymerase proteins that, together with the viral RNA segments, form the ribonucleoproteins (RNPs). The RNP genes form host-specific lineages and harbor host-associated genetic signatures. The functional significance of these determinants has been studied by reassortment and reverse genetics experiments, underlining the influence of the global genetic context. In some instances the molecular mechanisms have been approached, pointing to the importance of the polymerase activity and interaction with cellular host factors. Better knowledge of determinants of host restriction will allow monitoring of the pandemic potential
Mary, if I may, I would like to try and address part of your question. You said That seems to indicate that there would be more than simple natural random mutation and variation at work here but this is not needed. It makes it much more difficult to get from A (the three matched polymerase proteins in H1N1(2008)) to B (the much more virulent 1918 combination) but this can be achieved either by one extraordinarily low probability jump or by achieving several intermediary steps. The only real proviso is that the intermediary steps can not be so unfit, relative to the wild type, that they go extinct before the next mutation can occur. Recombination is not really a factor as it is only relevant to multiple changes within the same RNA strand, and we are discussing complementary changes across three strands which, once transcribed, form three proteins which then combine to perform one biological process (which makes it hardly surprising that fiddling with one part in isolation is not as effective as changing all three together as a matched set nor is it odd that the fourth player is the NP protein as this matched set is designed to interact with the NP part of this RNP). The probabilities are further reduced by the number of possible combinations that produce high virulence - which is an unknown variable. H5N1 is obviously even more virulent and has found a completely different solution to the problem; as we can not relate virulence to structure (tertiary protein structure) we have no way of knowing if there are millions of permutations or just a few nor can we say if in many of them the polymerase complex will be key. In addition to reveres comments about single base insertions any one or two (but not three) base insertion is going to change every amino acid down stream from that point which (if the insertion is near the start of the sequence) is likely to make the resultant protein once folded up so different to its normal design as to be very unlikely to be able to play its assigned biological role, ergo non functional viron. I am sure revere could have explained it better but I hope it helped.
e.g. take the 1918-segments 1,2,3,5,7,8
and 4,6 from the 1957 H2N2-pandemic.
that could be very virulent and transmissable and
most of us have no immunity
just feed the ferrets orally with lots of different
flu-viruses and whatch if some become infected.
Should be easy.
Should have been done.
If positive, they would have published ?!?
where is the thread with Tom and the Doctor and Mary and
Anon, that particular discussion -- amongst others -- @ EM -- Bird flu: 'tis the season (December 19, 2008) by revere
Historical Facts Associated With Influenza:
The last influenza pandemic occurred nearly 100 years ago, and this deadly outbreak resulted in about 50 million deaths worldwide. Influenza is caused by a virus, which is a parasite that needs a host to survive and reproduce.
It was called the Spanish Flu because the first human case was identified there. The pandemic ended up killing more than those that died during WWI. Understandably there was panic among people worldwide, as influenza was not discovered until 1933, so the mystery was rather frightening of what was happening.
Those who survived have allowed others to obtain antibodies from them to develop other antibodies for future viral outbreaks that may occur with this type of virus. This last influenza pandemic also allowed others to obtain this virus from those who died as a result to facilitate effective treatments and vaccines for viral outbreaks that may happen in the future as well.
The virus responsible for the 1918 pandemic was an avian influenza. Nearly 700,000 people in the U.S. died as the result of the Spanish Flu- and those that did die was due often to a bacterial pneumonia that followed the viral invasion and damage. Ultimately, this pandemic killed nearly 3 percent of humans infected. Normally, an influenza strain may kill less than one percent of those infected. The Spanish Flu caused an unusually severe immune response in the human host which made it very deadly due to overkill of the cells of this host.
The influenza viruses are categorized as A, B, and C. The Influenza A virus is the one that historically has caused pandemics that have developed-, such as the Spanish Flu Pandemic. The other influenza pandemics primarily have occurred in countries in Asia.
With influenza, it is understood that the disease influenza is a disease caused by a RNA virus that can infect both mammals and birds. In fact, this particular virus can mutate to where it can be shared between the two life forms and multiply within each one of them. Unlike coryza, influenza expresses symptoms more severely, and usually lasts two weeks until one recovers who has the flu. Influenza, however, poses a danger to some with compromised immune systems, such as the chronically ill, so the recommendation is greater in such populations, along with women who may be pregnant during the flu season, residents of nursing homes or chronic care facilities. Health care personnel are encouraged to get the flu vaccine as well. Such populations allow influenza to progress to deadly pneumonia.
Symptoms of influenza usually start to express themselves symptomatically 36 hours after being infected with the virus. Over 10 percent of the population is infected with this virus every year- resulting in about 200,000 hospitalizations and nearly 40,000 deaths. This seasons first influenza case was identified in Delaware in November of 2008, and it was a type B influenza strain.
The flu vaccination contains three viral strains of suspected viruses for flu outbreaks during a particular winter season, as determined by the World Health Organization, as well as the Centers for Disease Control, and other organizations. Unfortunately, the influenza vaccine administered last flu season was largely ineffective due to unsuspected strains of the virus infecting others, although about 140 million injections of this vaccine were administered.
After giving the vaccination dose to one, it takes about 10 days for that person to build up the immunity for the disease of influenza. The months of October to December are recommended to receive this vaccine. And the vaccine is about 50 percent effective in offering protection from influenza, according to others. Vaccines are a catalyst for antibody production in humans, which protect them against the virus. Influenza vaccines can be given by injection or nasally.
Anti-virals, on the other hand, decrease greatly the ability for viruses to reproduce once established in a human.
The Avian influenza that many have heard of is potentially the next flu pandemic- as humans have no immunity to what is called the H5N1 virus- on of about 1 strains of avian Influenza. For an Influenza pandemic to occur, which means a global disease existence, the virus must emerge from another species to humans without immunity, as well as the ability to make more humans ill than normal. Also, the virus must be highly contagious for a pandemic to occur. The H5N1 virus appears to replicate in the human GI tract and also has a longer incubation period in humans, one to two weeks, compared with other influenza strains. The H5N1 Avian influenza virus seems to have become progressively more pathogenic in the past decade, according to others.
With the Avian Influenza existing with the H5N1 strain, millions of birds have been slaughtered due to the danger and unpredictability of this strain. The first human case infected with this strain occurred in China in 1997. The first human avian flu case outside of China was identified in 2003 in the Netherlands. The first recorded incidence of human-to-human transmission of the H5N1 virus was in Thailand in 2004. In 2006, it was discovered that the H5N1 had split into two separate strains. There have been outbreaks of Avian flu in about 15 countries in the world so far- with Indonesia being the worst. Migratory birds spread this influenza virus between continents.
The pathogenic strength of the H5N1 strain varies due to constant re-assortment or switching of genetic material between the viruses- essentially creating a hybrid of what it was before this occurs. So far, about 300 people worldwide have been infected with this strain- and about half have died from the infection. Vaccinations are being developed and reformulated constantly at this time due to the pandemic threat of the H5N1 Influenza virus.
Yet, the normal flu season that is now occurring was supplied with 150 million vaccines in the United States. However, some studies have shown that this vaccine is rather ineffective based on incidences of the acquisition of the influenza virus by others anyway.
The influenza season peaks between the months of January and March. The vaccine for this influenza season is manufactured by 6 different companies. Yet the strains chosen are speculated influenza viruses, as this does not eliminate the chance of a new and dominant influenza viral strain that possibly could cause a pandemic. It takes manufacturers about 6 months to make and formulate the influenza vaccination. There is a vaccine for this illness that is produced every year according to which type of virus may be prevalent during a particular flu season. The presence of influenza can be widespread in certain states, yet not others. The vaccination is recommended to be administered to those who are at high risk, such as the chronically ill. Also, it is recommended that those under 18 years of age get the vaccine, as well as those people over the age of 50. Furthermore, those people who regularly take aspirin should receive the vaccine, as the influenza disease can become a catalyst for what is called Reyes Syndrome. Pregnant women should receive the vaccine as well- as there are many other vaccines available to fortunately prevent other diseases, perhaps.
Dan: Do you read this blog at all? We have done over a thousand posts on various aspects of this. Some of your "historical" facts aren't even correct. I'm not sure what your objective is.
I recall Baxter Pharma had a mix up recently. Somehow the wrong ingredients for the "standard" flu vaccine got sent to labs throughout Europe. The alarm bells went off when the ferrets they were testing, died. Makes me wonder if the variant made it to the population. Baxter also has a lab about 50 miles from Mexico City.
Great site by the way!
1. So how does the new H1N1 (swinw flu) compare to the 1918 H1N1 with respect to the mentioned "virulence markers" (PB1,PB2,PA) then ?
2. As even the 1918 flu was not more severe than "normal" flu in the vast majority of cases (given a 3% CFR, the disease must have been harmless & self-limiting in 97% of all cases given the absence of ventilators, antibiotics, antivirals) So shouldn't we look for the main factor responsible for the severity of the 3% severe cases to be in the host rather than in the virus ?
h1n1: Swine flu doesn't have any of them. But remember, virulence is a combination of host, environment and virus, not any one of them.
really this is very exllent work and article