Influenza A/Mexico/2009 (H1N1) update

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

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

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

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

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

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?

Swine influenza A/Mexico/2009 (H1N1) update

reassortment-swineHere is an update on the global swine flu situation as of 29 April 2009.

There are now 257 laboratory confirmed cases, with 7 deaths, in 11 countries. In the US there are 109 cases  in 11 states. There are many more suspected cases; together the statistics indicate widespread dissemination of the new H1N1 influenza virus. I no longer doubt that this is the next pandemic strain. WHO will probably soon raise the level of influenza pandemic alert from phase 5 to phase 6. Important questions include whether spread will continue in the northern hemisphere through the summer, or stop very soon, as is the case with most influenza virus outbreaks. Unfortunately the southern hemisphere seems in for an extended flu season. Will antivirals be useful in reducing morbidity and mortality? Will the virus returns to the north in a more virulent form in the fall? Can a vaccine be prepared in time?

Viral RNA sequences from 12 new isolates were deposited at NCBI, bringing the total to 32. Conspicuously missing are sequences from Mexican isolates. In a Science Magazine interview, Ruben Donis, Chief of the molecular virology and vaccines branch at CDC, indicated that strains from Mexico and elsewhere are “very, very similar. Many genes are identical. In the eight or nine viruses we’ve sequenced, there is nothing different.” It’s still not clear why these sequences have not been released; clearly the work has been done. In any case, his statement confirms what we have suspected from examining other isolates, that the Mexican strains are not sufficiently different to explain their apparent higher pathogenicity.

I highly recommend reading the interview with Dr. Donis, as it contains a wealth of information about the new H1N1 virus. Much of it will be difficult to understand for those without familiarity with influenza virus, but you can send your questions to virology blog. One interesting aspect concerns the statement last week that the new virus was composed of genes from pig, human, and avian sources. Examination of the sequences in the past week has revealed the virus to be composed of RNAs solely from swine viruses. Here is what Dr. Donis said:

Q: Is it of swine origin?

R.D.: Definitely. It’s almost equidistant to swine viruses from the United States and Eurasia. And it’s a lonely branch there. It doesn’t have any close relatives.

Q: So where are avian and human sequences?

R.D.: We have to step back [to] 10 years ago. In 1998, actually, Chris Olsen is one of the first that saw it, and we saw the same in a virus from Nebraska and Richard Webby and Robert Webster in Memphis saw it, too. There were unprecedented outbreaks of influenza in the swine population. It was an H3.

The PB1 gene, that was human. H3 and N2 also were human. The PA and PB2, the two polymerase genes, were of avian flu. The rest were typical North American swine viruses. Those strains were the so-called triple reassortants.

I’ll post an entry this weekend on the history of swine viruses, which should help clarify Dr. Donis’ explanation.

On Friday, 1 May I will be holding a FAQ session on swine flu with Marc Pelletier of Futures in Biotech. A video stream of our conversation will be broadcast live at 4:00 PM EST at live.twit.tv. Send us your questions via twitter to @profvrr.

Oseltamivir resistance decreases influenza aerosol transmission

tamifluThe isolates of influenza virus obtained in the current global outbreak have proven to be resistant to the adamantane antivirals, but susceptible to oseltamivir (Tamiflu) and zanamivir (Relenza). Consequently the two neuraminidase inhibitors will likely be used extensively to control the outbreak until a vaccine is available. Extended use of the antiviral drugs will undoubtedly lead to the selection of drug resistant influenza virus mutants. However, there might be a silver lining to this otherwise dismal prediction.

Transmission of oseltamivir-resistant influenza viruses was studied in a guinea pig model for infection. Viruses with either one or two amino acid changes that lead to drug resistance multiplied normally in guinea pigs, but the viruses were transmitted very poorly to other animals by aerosols. Oseltamivir-sensitive viruses were transmitted efficiently by aerosol among animals. Interestingly, both oseltamivir-sensitive and oseltamivir-resistant viruses transmitted efficiently by the contact route among guinea pigs placed in the same cage.

These observations indicate that mutations that cause resistance to oseltamivir reduce aerosol, but not contact transmission of influenza virus. The authors speculate that the amino acid changes associated with drug resistance also decrease the enzymatic activity of the viral NA (neuraminidase) protein. One of the functions of this viral protein is to facilitate release of newly synthesized virus particles from the surface of the infected cell. Mutations that cause drug resistance may lead to impaired virus release, and therefore poor transmission by the aerosol route.

Although these results are encouraging, it is not clear if drug resistance mutations would have a similar effect on aerosol transmission of the current A/Mexico/2009 (H1N1) influenza viruses. A high proportion of the influenza H1N1 viruses from the previous season were resistant to the drug, yet the viruses spread extensively. One possible explanation for the discrepancy is that influenza in guinea pigs does not lead to coughing or sneezing. Hence, the virus-laden aerosols produced by guinea pigs are a consequence of normal breathing. In humans, air turbulence caused by coughing and sneezing might be sufficient to dislodge virus particles that in the guinea pig would remain firmly attached to the epithelial surface. Nevertheless, the results suggest that simple hygienic measures could slow the spread of a drug resistant virus that transmits by efficiently by contact and poorly by aerosol.

Bouvier, N., Lowen, A., & Palese, P. (2008). Oseltamivir-Resistant Influenza A Viruses Are Transmitted Efficiently among Guinea Pigs by Direct Contact but Not by Aerosol Journal of Virology, 82 (20), 10052-10058 DOI: 10.1128/JVI.01226-08

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.

Influenza virus transmission

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