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swine influenza

Influenza A/Mexico/2009 (H1N1) update

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There are some interesting statistics on influenza in the current issue of CDC’s Morbidity and Mortality Weekly Report. The first is this map showing the number of confirmed cases (N = 1,882) of novel influenza A (H1N1) virus infection worldwide as of May 6, 2009:

m817a1f3

With the exception of a few countries, the vast majority of confirmed cases have been in the northern hemisphere. I suspect this situation will change rapidly – flu season is winding down in the northern hemisphere, and is about to begin in the south. The fact that there are already confirmed cases in the southern hemisphere means the virus is already there, and likely to spread further. CDC says as much, although with considerably more uncertainty:

Summertime influenza outbreaks in temperate climates have been reported in closed communities such as prisons, nursing homes, cruise ships, and other settings with close contact. Such outbreaks typically do not result in community-wide transmission, but they can be important indicators of viruses likely to circulate in the upcoming influenza season. The novel influenza A (H1N1) virus has been circulating in North America largely after the peak influenza transmission season. For that reason, the epidemiology and severity of the upcoming influenza season in the southern hemisphere or in the northern hemisphere cannot be predicted. The imminent onset of the season for influenza virus transmission in the southern hemisphere, coupled with detection of confirmed cases in several countries in the southern zone, raise concern that spread of novel influenza A (H1N1) virus might result in large-scale outbreaks during upcoming months. Countries in the southern hemisphere that are entering the influenza season should anticipate outbreaks and enhance surveillance accordingly. Influenza virus can circulate year round in tropical regions; therefore, these countries should maintain enhanced surveillance for novel influenza A (H1N1) virus.

Also shown in bar graph form are the number of confirmed (N = 822) and suspected (N = 11,356) cases of novel influenza A (H1N1) virus infection, by date of illness onset in Mexico, from March 11 – May 3, 2009:

m817a1f1

These data suggest several interesting possibilities. Infection with the new H1N1 strain might have begun as early as March 11, although the early cases are suspected, not laboratory confirmed. After a period of relatively few infections, the number of cases rapidly climbed to a peak and then quickly declined. Of course, new outbreaks are still possible. If we take the number of deaths in Mexico (42) and divide them by the number of laboratory confirmed cases (949), the mortality rate is 4.4% – higher than the 2.5% observed during the 1918-19 pandemic. However, if we divide the number of deaths by the number of suspected cases (11,932), the fatality rate is more in line with typical seasonal influenza – 0.4%. Which number is correct awaits determination of the actual number of cases.

It is also informative to examine CDC’s case definition for influenza – fever plus cough or sore throat. This definition is very broad and could easily include infections caused by other respiratory viruses, such as rhinoviruses, coronaviruses, adenoviruses, and paramyxoviruses. Indeed, I have heard that some of the suspected cases of H1N1 influenza in New York are in fact a consequence of rhinovirus infection. Analyzing an outbreak by using the suspected number of cases is dangerous – akin to a scientist ‘wasting clean thoughts on dirty data’.

Why swine flu isn’t so scary

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Peter Palese has written an excellent opinion piece for the Wall Street Journal on why swine flu is not that scary. His arguments may bring some comfort for those readers of virology blog who are worried about the impact of the new influenza H1N1 strain. Even if you are not worried, the scientific basis for his arguments are compelling and answer some of the many questions I have been receiving in the past week. I can’t think of anyone’s opinion on influenza virus that I value more – and it’s not just because I did my Ph.D. research in his laboratory. He’s an outstanding scientist with balanced, well-informed opinions. Herewith are some excerpts from his piece.

First, Palese reviews the concerns about the new H1N1 viruses:

1. The swine virus belongs to the same H1N1 group as did the 1918 pandemic virus.
2. The swine virus is readily transmitted from human to human.  At this point, swine virus isolates have been reported on four continents.  The avian H5N1 virus (another virus with pandemic potential) was never proven to readily transmit from person to person; rather, humans were probably infected directly from chickens and these infections required large quantities of virus.
3. The swine virus shows an unusual robustness in emerging outside the normal seasonal period for the virus.  Influenza viruses are rarely isolated at the end of April in the northern hemisphere, and winter hasn’t yet started in New Zealand, where several isolates have already been reported.
4. Mutations and/or acquisition of genes derived from other human or animal influenza viruses could make the swine virus into something much more virulent than it is now.  Mutations and acquisition of genes are natural processes for influenza viruses against which there are no man-made interventions.  Furthermore, these processes (and the extent to which they could enhance virulence) cannot be predicted.

Next, he argues why we should be optimistic:

1. In 1976 there was a an outbreak of an H1N1 swine virus in Fort Dix, New Jersey, which showed human to human transmission but did not go on to become a highly virulent pandemic strain.
2. The presently circulating swine virus is most likely not more virulent than the other seasonal strains we have experienced over the last several years.
3. The current swine virus lacks an important molecular signature (the protein PB1-F2) which was present in the 1918 virus and in the highly lethal H5N1 chicken viruses.  If this virulence marker is necessary for an influenza virus to become highly pathogenic in humans or in chickens, then the current swine virus doesn’t have what it takes to become a major killer.
4. Since people have been exposed to H1N1 viruses over many decades, we likely have some cross-reactive immunity against the swine H1N1 virus. While it may not be sufficient to prevent becoming ill, it may very well dampen the impact of the virus on mortality.  I would postulate that by virtue of this “herd immunity” even a 1918-like H1N1 virus could never have the horrific effect it had in the past.  The most likely outcome is that the current swine virus will become another (fourth) strain of regular seasonal influenza.
5. The landscape of vaccines and anti-influenza drugs has dramatically improved over what it was just a few years ago.  Based on what we know of the structure and sequence of the swine virus, these FDA-approved drugs and FDA-licensed vaccines (modified to include the swine strain) would be highly effective against this new virus.  Also, present technologies as well as manufacturing capacities will allow us to make sufficient quantities of a swine virus vaccine for the winter 2009-10 season in this country.

In closing, he notes that we have a vastly improved infrastructure to deal with novel emerging diseases:

The preparedness plans developed against the H5N1 influenza threat dramatically improved overall surveillance (we would probably not have learned so fast about the swine virus were it not for these improved capabilities).  Major advances have been initiated by our government to develop new and improved manufacturing processes and exciting new vaccine and antiviral approaches are also in the pipeline, and they show promise of tipping the balance in favor of humans against a devious virus.  For example, universal influenza vaccines (one long-lasting vaccine against all strains) and broadband antivirals are being developed in our academic laboratories and in innovative small biotech companies.  This work has been primarily funded by the NIH and the CDC and it will pay off by diminishing the future impact of influenza on the health of our citizens and on the economy of our country.  It is prudent to prepare against swine influenza, but equally important to keep a balanced outlook and an awareness of our current capabilities.

Coming from such a well-informed and experienced source, these arguments are compelling. Please pass them on to anyone you know who might be worried by the recent emergence of the new influenza H1N1 virus.

Influenza A/Mexico/2009 (H1N1): Absence of crucial virulence marker

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influenza-rna-2The second RNA segment of the influenza virus genome encodes two proteins, PB1 and PB1-F2.  The latter protein is believed to be an important determinant of virulence of influenza virus. Can we learn anything about the virulence of the new influenza virus H1N1 strains from a study of this protein?

During influenza virus infection, PB1-F2 is targeted to the mitochondria, where it induces a form of cell death known as apoptosis. Experiments in a mouse model of influenza virus infection have shown that PB1-F2 regulates lethality of the virus. By comparing the infection of mice with two strains of influenza virus, one of which produces much lower levels of the PB1-F2 protein, it was found that the protein enhances inflammation and increases frequency and severity of secondary bacterial pneumonia. A specific amino acid at position 66 of this protein appears to be an important determinant of viral virulence. This amino acid is a serine in the 1918 H1N1 influenza virus, in a 1997 avian H5N1 isolate from the Hong Kong outbreak, and in the H2N2 (1957) and H3N2 (1968) pandemic strains. Other less pathogenic influenza virus isolates have an asparagine at this position. Two viruses were constructed which differ at amino acid 66 of the PB1-F2 protein, and the virulence of these viruses was determined in mice. The influenza virus with a serine at amino acid 66 was pathogenic in mice, while the virus with an asparagine was significantly less virulent. Increased pathogenicity of the virulent virus was associated with higher levels of virus replication in the lungs. The results of these studies show that the PB1-F2 protein affects pathogenicity in a mouse model, and that position 66 plays an important role.

h1n1-pb2-f2
Truncated PB1-F2

Because the amino acid change N66S of PB1-F2 is present in the three previous pandemic influenza virus strains – 1918 H1N1, 1957 H2N2, and 1968 H3N2 – it would be of interest to determine which amino acid, N or S, is present in the new H1N1 influenza virus strain that is spreading globally. However, examination of the nucleotide sequence of RNA from the current H1N1 isolates shows that these viruses do not even produce a PB1-F2 protein – a stop codon is present after amino acid 11 (see figure). In fact, many other influenza virus strains do not produce the protein. While the PB1-F2 protein is not the only determinant of influenza virus virulence, we can at least eliminate any contribution of this viral protein to increased lethality. As Peter Palese has written in today’s Wall Street Journal, “If this virulence marker is necessary for an influenza virus to become highly pathogenic in humans or in chickens, then the current swine virus doesn’t have what it takes to become a major killer.”

Conenello, G., Zamarin, D., Perrone, L., Tumpey, T., & Palese, P. (2007). A Single Mutation in the PB1-F2 of H5N1 (HK/97) and 1918 Influenza A Viruses Contributes to Increased Virulence PLoS Pathogens, 3 (10) DOI: 10.1371/journal.ppat.0030141

MCAULEY, J., HORNUNG, F., BOYD, K., SMITH, A., MCKEON, R., BENNINK, J., YEWDELL, J., & MCCULLERS, J. (2007). Expression of the 1918 Influenza A Virus PB1-F2 Enhances the Pathogenesis of Viral and Secondary Bacterial Pneumonia Cell Host & Microbe, 2 (4), 240-249 DOI: 10.1016/j.chom.2007.09.001

Influenza virus RNA: Translation into protein

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influenza-rna-2
figure 1

Let’s resume our discussion of the influenza virus genome. Last time we established that there are eight negative-stranded RNAs within the influenza virion, each coding for one or two proteins. Now we’ll consider how proteins are made from these RNAs.

Figure 1 shows influenza RNA segment 2, which encodes two proteins: PB1 and PB1-F2. The (-) strand viral RNA is copied to form a (+) strand mRNA, which in turn is used as a template for protein synthesis. Figure 2 (below) shows the nucleotide sequence of the first 180 bases of this mRNA.

The top line, mostly in small letters, is the nucleotide sequence of the viral mRNA. During translation this sequence is read in triplets, each of which specifies an amino acid (the one-letter code for amino acids is used here). Translation usually begins with an ATG which specifies the amino acid methionine; the next triplet, gat, specifies aspartic acid, and so on. Only the first 60 amino acids of the PB1 protein are shown; the protein contains a total of 758 amino acids.

Most of the influenza viral RNAs code for only one protein. However, RNA 2 (and two other RNAs) code for two proteins. In the case of RNA 2, the second protein is made by translation of what is known as an overlapping reading frame.

On the second line of the RNA sequence in figure 2 is an atg highlighted in red. You can see that this atg is not in the reading frame of the PB1 protein. However, it is the start codon for the second protein encoded in RNA 2, the PB1-F2 protein (F2 stands for frame 2, because the protein is translated from the second open reading frame). Figure 3 shows how PB1-F2 is translated. The sequence of the viral RNA is shown from the beginning, except that reading frame 1, which begins at the first ATG, is not translated. Rather, we have begun translation with the internal atg, which is in the second reading frame. This open reading frame encodes the PB1-F2 protein which, in this case, is 90 amino acids in length (its length varies in different isolates). The protein is much shorter than PB1 because translation stops at a termination codon (tga) long before the end of the RNA. Because PB1-F2 is encoded in reading frame 2, its amino acid sequence is completely different from that of PB1.

figure 2
figure 2
figure 3
figure 3

The sequences used for this example are from the 1918 H1N1 strain of influenza. Notice the amino acid of PB1-F2 which is highlighted in blue. This amino acid has an important role in the biological function of the protein, which we will consider in a future post.

My apologies if the figures and text are not optimally aligned. A blog post is not the optimal format for such information, but in the interest of time I have not explored other options. Suggestions for improvement are welcome.

Send your questions to virology@virology.ws.

Influenza A/Mexico/2009 (H1N1): Questions and answers

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questionHere are my answers to questions about the currently circulating influenza H1N1 strain (formerly swine flu) sent by readers of virology blog.

A reader from Mexico shared the following numbers with virology blog, then asked a question about hog cholera.

Q: There are 312 confirmed cases of swine virus here in Mexico and 12 of them have died. This means 3.8%. In the rest of the world there are 159 confirmed cases of swine virus infection, but no deaths. It gets even worse, there have been reported 176 deaths by pneumonia in this past two weeks, but only 12 are positive for the swine virus, this means that only 6.8% of the fatalities can be blamed on the swine virus. Three states San Luis Potosi, Oaxaca and Aguascalientes have together reported 195 cases of pneumonia with 24 deaths, that is 12.3% die. While three other states (Tlaxcala, Veracruz and Coahuila) have together reported 154 cases of pneumonia with no deaths…(a similar situation as the rest of the world).

I have read that hog cholera can increase the death toll of influenza virus above 10%. What is hog cholera and why does this worsen flu?

A: Hog cholera is an infectious disease of pigs caused by a virus known as classical swine fever virus. Infection with this virus leads to fever, skin lesions, convulsions and often death. It is also immunosuppressive, therefore worsening influenza in swine.

Q: How risky is it to travel internationally (to places other than Mexico) at this time? How likely is one to pick up a flu on an airplane, in an airport, while staying at a hotel or hostel, etc? I am supposed to be visiting Spain for a month – my travel insurance company says they won’t cover any cancellation costs because no one has issued a travel advisory against Spain. Does this mean that it is more or less safe to visit Spain, although there are cases reported there as well? I am confused. How likely is one to become sick while travelling? Would it be better to stay home at this point?

A:At this point the new influenza virus strain seems to be nearly everywhere, even in the US, based on suspected cases. Therefore travel poses no increased risk. However, travel is a good way to become infected – airports, hotels, airplane, are all good venues for transmission by aerosol or contact (see an earlier virology blog post on transmission – the airplane transmission story is particularly interesting). So in my view, you probably have a higher risk of being infected because you will be in contact with more people while traveling than if you stayed at home. But if you take precautions like cleaning hands with an alcohol-based cleaner, refrain from touching mouth/eyes/nose, you can minimize the likelihood of exposure. I doubt there will be travel advisories issued for Spain because by the time there are many cases there, the virus will be elsewhere as well. If your trip is not imminent, you can delay and make a decision later.

Q: Is Vitamin D effective against influenza? And does this also help explain seasonality?

A: It has been hypothesized that vitamin D was effective against influenza (see this paper), but this has never been rigorously proven. Seasonality better correlates with temperature and humidity.

Q: Your note that CDC does not recommend use of face masks for reducing viral spread is somewhat inaccurate. In addition, the posted article titled “Influenza Viral Transmission” does not take transmission through contact with GI substances into account. In CDC’s Interim Recommendations for Facemask and Respirator Use in Certain Community Settings Where Swine Influenza A (H!N!) Virus Transmission has been detected, it is stated, “When crowded settings and close contact with others cannot be avoided, the use of facemasks or respirators in areas where transmission of swine influenza A (H1N1) virus has been confirmed should be considered. The documents then gives three circumstances.

A: Thank you for your comments and support. I understand that in some cases facemasks/respirators are recommended. As I pointed out in virology blog, on the front page of CDC, which most laypersons will see, there is no recommendation for the use of such protection. As you know, when not use properly, face masks do not function as intended and most laypersons will not have the opportunity to use respirators (by layperson I mean someone not involved in health care). As for GI spread, influenza cannot and does not replicate in the GI tract of humans (although it does so in birds). Concurrent infections with other pathogens (e.g. norovirus) may lead to GI symptoms.

Q: Our elite team of news reporters (who still refer to this as Swine flu) listed vomiting and diarrhea as symptoms. This seems peculiar for flu. During the regular flu season, I remember telling patients to look out for fever, arthralgias, cough etc…never anything about vomiting and diarrhea. Is this a legitimate symptom, or just the media being as vague as possible?

A: Influenza cannot and does not replicate in the GI tract of humans (although it does so in birds). Concurrent infections with other pathogens (e.g. norovirus) may lead to GI symptoms.

Q: ALL OF US WILL GOING TO BE IN CONTACT WITH THE VIRUS  IN THE NEXT MONTHS OR YEARS WHAT IS THE BEST TO GET SICK NOW OR LATER I THINK WE MUST RESIST ALL WE CAN BECAUSE I THINK ON DECEMBER THE VACCINE COULD BE DISPONIBLE.

A: Do not attempt to become infected now; the outcome of influenza infection may not be benign.

Q: I’d like to know the potential lethality of swine influenza  as compared with other types of influenza viruses. IS THERE ANY REFERENCE ABOUT THE RISK OF INFECTION IN OPEN OR CLOSED SPACES??

A: There is no reason to believe that the current H1N1 virus, which originated in swine, is any more lethal than any other influenza virus, with the exception of avian H5N1 strains and the 1918-19 strain. Transmission is likely to be more efficient in closed spaces (such as an airline cabin; see this post).

Q: How long would you expect human immunity to exist within an individual infected with H1N1 virus?

A: I would expect immunity to be lifelong against that specific strain. However, immunity will not completely protect agains future antigenically drifted viruses of the same lineage.

Q: One small correction. N-95s are respirators, not face masks. Respirators must meet specifications for filtering out specific types and sizes of particles. An N-95 is a particulate respirator that when fitted properly filters 95% of particles 0.3 microns or larger. Face masks generally refer to medical and surgical masks that need only provide a physical barrier.

Thank you.

Q: At the end of a recent interview with Science magazine, Dr. Donis of the CDC states that the USA has received 300 samples of this virus from Mexico. Why has the CDC failed to publish the sequences of any of these 300 Mexican samples Dr. Donis says have been received from Mexico? Doesn’t this seem to be an intentional act?

A: I’m not sure. It’s been suggested that the Mexican government wants to keep rights to the use of the viruses as a vaccine and therefore do not want the sequence released. I suspect we’ll find out in the coming week.

Q: Regarding Dr. Schuchat’s statement, could it be that if you trace the lineage of certain viral genome segments that their origin could be avian, for example? i.e., perhaps the A/swine/Indiana/P12439/00 isolate was/is a reassortant originally containing some segments of avian origin? Just a thought, I have not personally looked closely at these sequences…

and also:

Q: But you still agree that this virus appears to contain genetic pieces from from four virus sources, correct? (one avian influenza viruses, one human influenza viruses, and two swine influenza viruses).

A: In 1998, recombinant viruses arose in pigs that were a combination of human, pig, and avian influenza virus RNAs. See Dr. Donis’ statement in yesterday’s Science interview. Today they look like swine viruses because they have been evolving in this animal for so many years.

Q: How do we account for the April 24 comments of Dr. Marie Gramer below, who has perhaps the largest library of swine flu virus isolates in the nation the day after Schuchat’s comments? “There have been no reports of this virus in pigs, said Dr. Marie Gramer, a swine flu expert with the University of Minnesota’s college of veterinary medicine. “It doesn’t seem to be very similar to anything that is currently circulating, from what I have,” said Gramer, who has an extensive library of swine flu virus isolates.”

A: I believe she is wrong.

Q: I do not understand why you are morally certain that the German and New York isolates arose from Mexico.

A: I am not certain, I only assume that because the New York and German tourists visited Mexico, that there were infected there and returned with the virus. However the point is moot if the Mexican viruses are very similar to all the others.

Q: Mexico’s chief epidemiologist insists this virus did not begin in a Mexican pig farm. “Miguel Angel Lezana, Mexico’s chief epidemiologist, told reporters…. the presence of Eurasian swine flu genes in the H1N1 virus makes it unlikely that the disease originated in a Mexican pig farm.”

A: Chief Epidemiologist? He should be fired. The fact that Eurasian swine flu genes are present in no way makes a Mexican origin more unlikely than any other.

Q: It’s taken me a while to digest the fact that the Mexican flu viruses haven’t been sequences. Shouldn’t this be bigger news? Why is it confined to a relatively obscure blog? (No offense, Dr. Racaniello.)

(I could not resist printing the preceding question. Not offended at all!)

Q: Canine influenza emerged a few years ago in Florida in greyhounds, and was found to have come from an equine influenza strain.  However, after entering the canine population, the virus is no longer able to infect horses.  Perhaps a similar thing is occurring here.

A: Swine are readily infected with human influenza viruses. So there is no reason to believe that the new human H1N1 virus would not be able to go back into pigs. We’ll see.

Q: Fucoidan is a sulfated polysaccharide primarily found in brown seaweeds.  Although the specific shape/sugar to sulfate compositions vary from seaweed to seaweed, the fucoidan used in this paper is from Undaria pinnatifida, one of the most popular dietary seaweeds consumed in Japan and Korea.  The sporophyll contains 8-12% fucoidan (the part of the Undaria used in this study, also called Mekabu in Japanese).  Is it possible that people in Japan will be protected from H1N1 if they eat Mekabu?

A: The concentrations of fucoidan used in the studies you provided are rather high and would not be present at such levels in Mekabu. Therefore eating the seaweed would not likely be of value in preventing infection.

Influenza virus RNA genome

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influenza_virus_rna

Within the influenza A virion are eight segments of viral RNA. These molecules carry the all the information needed to make new influenza virus particles. These eight RNAs are shown schematically as olive green lines at the top of the illustration. RNAs are chains of four different nucleotides, A, C, G, U. In the case of influenza virus, the eight RNAs are a total of about 14,000 nucleotides in length. The nucleotides make up the genetic code – it is read by the cell’s translation machinery in groups of three, with each triplet specifying an amino acid.

There are two important aspects of these viral RNA that we must consider. First, you can see that the ends of the RNAs are labeled 3′ and 5′. Nucleic acids have polarity, in that one end of the chain is chemically different from the other. Such polarity is represented by 5′ or 3′. The second point is that when a nucleic acid is copied, or duplicated, by enzymes called polymerases, a strand of the complementary polarity is produced. Influenza viral RNAs are called (-), or negative strand RNAs, because they are the opposite polarity of the RNA that is translated to make protein. The RNA molecules that are templates for the synthesis of proteins are defined as having having (+), or positive polarity. Upon entering the cell, the (-) strand influenza viral RNAs must be copied into complementary (+) strands, so that they can serve as templates for proteins. The viral RNAs are copied by an enzyme – called RNA polymerase – that is carried into the cell with the virus.

In the above scheme, the olive green lines are the (-) strand RNAs found in the influenza virion. Once the virion enters the cell, these 8 RNAs are copied into (+) strand mRNAs. Finally, the mRNAs can serve as templates for the synthesis of proteins. The specific viral proteins that are produced by each viral mRNA are shown at the bottom of the illustration. From this picture we see that, for example, RNA segment 4 codes for the viral HA protein, and RNA segment 6 codes for the viral NA protein. Note also that some RNA segments encode for more than one protein. Both influenza A and B viruses have 8 RNA segments, while the influenza C viruses have 7.

Influenza viruses are called (-) strand RNA viruses because of the polarity of the RNA that is carried in the virion. Other RNA viruses – such as poliovirus – are (+) strand RNA viruses, because their genomic RNA can be translated into protein immediately upon entering the cell.

Any questions before we proceed?