Transgenic pigs resistant to foot-and-mouth disease

Foot-and-mouth disease virus (FMDV) infects cloven-hoofed animals such as cattle, pigs, sheep, goats, and many wild species. The disease caused by this virus is a substantial problem for farmers because infected animals cannot be sold. Transgenic pigs have now been produced which express a short interfering RNA (siRNA) and consequently have reduced susceptibility to infection with FMDV.

FMDV is classified in the picornavirus family which also contains poliovirus and rhinoviruses. The virus is highly contagious and readily spreads long distances via wind currents, and among animals by aerosols and contact with farm equipment. Infection causes a high fever and blisters in the mouth and on the feet – hence the name of the disease. When outbreaks occur, they are economically devastating. The 2001 FMDV outbreak in the United Kingdom was stopped by mass slaughter of all animals surrounding the affected areas – an estimated 6,131,440 – in less than a year.

Vaccines against the virus can be protective but they are not an optimal solution. One problem is that antigenic variation of the virus may thwart protection. In addition, countries free of FMDV generally do not vaccinate because this practice would make the animals seropositive and prevent their export (it is not possible to differentiate between antibodies produced by natural infection versus immunization). Furthermore, if there were an outbreak of foot-and-mouth disease in such countries, the rapid replication and spread of the virus would make vaccination ineffective – hence culling of animals as described above is required. Clearly other means of protecting animals against FMDV are needed.

Synthetic short interfering RNAs (siRNA) have been shown to block viral replication in cell culture and in animals. To achieve such inhibition, short synthetic RNAs complementary to viral sequences are produced in cells. Upon infection, these siRNAs combine with the cellular RNA-induced silencing complex (RISC) which then targets the viral RNA for degradation.

To determine if siRNA could be used to protect pigs from foot-and-mouth disease, a complementary viral sequence was first identified that blocks FMDV replication in cell culture by ~97%. A vector containing this siRNA sequence was then used to produce transgenic pigs. Such animals not only express the antiviral siRNA, but as the encoding vector is present in germ cells, it is passed on to progeny pigs.

Expression of the siRNA was confirmed in a variety of transgenic pig tissues, including heart, lung, spleen, liver, kidney, and muscle. In fibroblasts produced from transgenic pigs, virus replication was reduced 30 fold. When transgenic pigs were inoculated intramuscularly with FMDV, none of the animals developed signs of disease such as fever or blisters of the feet and nose. In contrast, control non-transgenic pigs developed high fever and lesions. Viral RNA levels in the blood of transgenic pigs were 100-fold lower than in control animals. At 10 days post-infection no viral RNA was detected in heart, lung, spleen, liver, kidney, and muscle, while high levels were observed in these organs from non-transgenic controls.

These results show that siRNAs can protect transgenic pigs from FMDV induced disease. An important question that must be answered is whether transgenic pigs still contain enough virus to transmit infection to other animals. In addition, siRNAs are short – 21 nucleotides – and a mutation in the viral genome can block their inhibitory activity. Therefore it would be important to determine if mutations arise in the FMDV genome that lead to resistance to siRNAs.

Even if transgenic siRNA pigs do not transmit infection, and viral resistance does not arise, I am not sure that consumers are ready to accept such genetically modified animals.

TWiV 89: Where do viruses vacation?

Hosts: Vincent Racaniello and Alan Dove

On episode #89 of the podcast This Week in Virology, Vincent and Alan review recent findings on the association of the retrovirus XMRV with ME/CFS, reassortment of 2009 pandemic H1N1 influenza virus in swine, and where influenza viruses travel in the off-season.

Click the arrow above to play, or right-click to download TWiV #89 (56 MB .mp3, 78 minutes)

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TWiV #30: A/Mexico/4108/2009 (H1N1)

twiv_aa_2001On episode #30 of the podcast “This Week in Virology”, Vincent, Dick, Alan, and Hamish Young focus on the new H1N1 influenza virus, which originated in swine and is likely to be the next pandemic strain.

Click the arrow above to play, or right-click to download TWiV #30 or subscribe in iTunes or by email.

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.


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


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

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.


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.

Structure of influenza virus

influenza-virion3In this week’s discussion of swine flu A/Mexico/09 (H1N1), we have considered many aspects of influenza virus biology that might not be familiar to some readers of virology blog. I thought it might be useful to explain how the virus multiplies, how it infects us, and how we combat infection. Today we’ll start with the basic structure of influenza virus, illustrated above.

The influenza virion (as the infectious particle is called) is roughly spherical. It is an enveloped virus – that is, the outer layer is a lipid membrane which is taken from the host cell in which the virus multiplies. Inserted into the lipid membrane are ‘spikes’, which are proteins – actually glycoproteins, because they consist of protein linked to sugars – known as HA (hemagglutinin) and NA (neuraminidase). These are the proteins that determine the subtype of influenza virus (A/H1N1, for example). We’ll discuss later how the HA and NA are given subtype numbers. The HA and NA are important in the immune response against the virus; antibodies (proteins made by us to combat infection) against these spikes may protect against infection. The NA protein is the target of the antiviral drugs Relenza and Tamiflu. Also embedded in the lipid membrane is the M2 protein, which is the target of the antiviral adamantanes – amantadine and rimantadine.

Beneath the lipid membrane is a viral protein called M1, or matrix protein. This protein, which forms a shell, gives strength and rigidity to the lipid envelope. Within the interior of the virion are the viral RNAs – 8 of them for influenza A viruses. These are the genetic material of the virus; they code for one or two proteins. Each RNA segment, as they are called, consists of RNA joined with several proteins shown in the diagram: B1, PB2, PA, NP. These RNA segments are the genes of influenza virus. The interior of the virion also contains another protein called NEP.

This week, when we discussed the nucleotide sequence of swine influenza RNAs, we were referring to these RNA molecules. Tomorrow I’ll show you how each RNA codes for protein. This way it will be easier to understand the meaning of the swine flu virus sequences that were released this week.

Let me know if this type of explanation is useful, and if you would like me to continue.

Swine flu A/Mexico/2009 (H1N1): Questions and answers

questionHere are my answers to questions about swine flu sent by readers of virology blog:

Q: Am I missing something?  How can a summer pandemic be unprecedented?  You cited a pretty famous example of one.  In fact nearly all of your examples seem to have occurred partly or mostly “out of season”.

A: You are correct; it is true that there were cases of influenza over the summer of 1918, even in the northern hemisphere. These were mainly in troops; I believe this situation was anomalous due to massive troop movements. But in 1968 the seasonal pattern was clear.

Q: I haven’t seen anyone address death rates for cases treated with antivirals vs. cases not treated with antivirals. For that matter, reporting of the cases in the US has been rather unclear about the extent to which confirmed and suspected cases are being treated with antivirals. Couldn’t it be that Mexico has only relatively recently started to consistently treat suspected cases with antivirals within the short window of effectiveness, and that most US cases have received treatment? This would explain a leveling off of the increase in number of deaths in Mexico and the “mild” cases we’re seeing in the U.S.  If this is not made clear, the public will mistakenly believe this flu to be benign and will not take the appropriate steps to mitigate its spread. And couldn’t this turn political as countries have different incentives to shut down their economies with quarantines and bans on public gatherings given their respective antiviral stockpiles?

A: I don’t have any information on the extent of use of antivirals anywhere. There are still so few confirmed cases that it would be premature to speculate, although it’s a good point.

Q: How are influenza viruses classified as H1, H2, etc? I had always thought it was serological, but that would imply that the seasonal H1N1 vaccine might have some efficacy against this H1N1 “swine” virus. Is there a more modern method for classification based on nucleotide sequence?

A: The subtyping (H1, H2, etc) is done by serology, using a panel of antibodies against the 17 known HA subtypes. This would imply some cross-reactivity among the H1 HAs of the swine flu strain and the previous H1N1 strain. Such cross-reactivity might confer perhaps not protection against infection but could lead to milder disease. CDC asserts that “Vaccination with seasonal influenza vaccine containing human influenza A (H1N1) would not be expected to provide protection against swine influenza A (H1N1) viruses” but it could reduce disease severity.

Q: I found these statistics on the newspaper “Reforma” so I have added the population for each state (in thousands) and calculated morbidity and mortality by adding all up (deaths, proven cases and probable (maybe) cases) and it turns out that the states with the highest morbidity do not have the highest mortality… for instance Tlaxcala has the highest morbidity for such a small state and San Luis Potosi has the highest mortality with a much lower morbidity….Could be that we are dealing with two different types of H1N1 viruses? one is the California that is not too virulent and a mutated one that is highly virulent and is not spreading that much….

A: These are certainly possible scenarios. Until we have sequences from the Mexican isolates we won’t know the answers. I do think the absence of sequence information from Mexico is exacerbating the panic.

Q: What are the chances of swine flu developing resistance against Tamiflu and other currently available antiviral drugs ?  Do you think that the distribution of Tamiflu and other drugs over the course of the last few years have been a mistake that leaves us less well-prepared in front of the current epidemic ?

A: The chance of swine flu becoming resistant to Tamiflu is very high; last season a high proportion of all circulating H1N1 viruses were resistant to the drug. Remember, the current H1N1 viruses are already resistant to adamantanes. I don’t think distribution of the drugs was a mistake; their use probably saved lives. What is a mistake is not having a deeper arsenal of antiviral drugs. We need to have at least 6 drugs for an effective antiviral approach.

Q: I live in Mexico and we are very confused here about the measures the government is taking against the flu. I have heard that it is not possible in the first 7 days passing the virus person-person, but only in the moment that the symptoms are already visible. Is that true?

A: Peak virus shedding occurs about a day before peak symptoms. I would not say it is not impossible to shed virus without symptoms, but in most cases, shedding is quickly followed by symptoms.

Q: I read somewhere that this new strain is one that contains parts of the H5N1 virus (bird flu) and other flues, including the H1N1 strain (human), and some genetic material from swine flu, but they’re just calling it the H1N1 virus. This is all very confusing, because that would have had to be genetically engineered in a lab and then released into the general public. They (the media) is also saying that it is a rapidly mutating virus (RNA, obviously, because they are unstable). Could this be genetically engineered? If someone could explain, that would be great.

A: Pigs are infected with many different influenza virus subtypes (H1N1, H3N2) and viruses with different sets of RNA segments can emerge. It does not have to be engineered in a lab to have RNAs from so many different viruses. This is not a virus anyone has seen before, and no scientist is smart enough to have made it so that it would be transmitted among humans.

We receive quite a few anxious emails from Mexico. Following is an example:

Q: I am living in Baja, Mèxico. This  swine influenza issue is getting really scary. And I suspect authorities  give one information then they give contradictory information, I think they don´t want people to panic. As we already are anyway.

a) La Paz, is really  hot weathered, unfortunately… and the hot weather hasn`t come yet, do you think hot weather would help to attenuate the spreading of the virus?

A: Yes, hot weather should interfere with aerosol transmission. But it is possible that the virus might continue to spread through contact.

b) How long do you think it would take to make a vaccine?

Six months. I have read that some companies feel it can be done faster, but the vaccines we have used successfully in the past require six months.

c) Now it is really difficult to find the Relenza or Tamiflu, but I have heard that some natural products sometimes help reinforced the inmunological system , like the onion, would you recommend to eat something in particular?

I am not aware of natural antiviral sources, but I would caution you against using natural products that might contain other dangerous materials (not onions, of course).

Q: I was reading that some countries do not have the techniques for testing if a virus is of the present H1N1 type. Which exactly is the procedure for this test and why it is so complex/expensive?

A: It is true that some of the assays are complex, and several are used. One method is to isolate the virus in cell culture by inoculating a nasal swab or throat wash specimen into cultured cells. Then a panel of antibodies against the 16 known HA subtypes are used in a variety of assays, such as neutralization assays, in which the capacity of the antibody to block infection is determined. For example, if the virus is an H1 virus, only antibodies against the H1 HA would block infectivity. Other diagnostic methods include direct fluorescent antibody testing, immunoassays, real-time reverse transcription-polymerase chain reaction, nucleotide sequencing, and multiplex reverse transcriptase-polymerase chain reaction. Some of these assays can be done directly on the nasal swab or throat wash. Viral culture is the “gold standard” for typing and subtyping of influenza viruses, but takes 3 to 7 days to culture the virus. In experienced hands they are not complex, but they must be done properly to have confidence in them. Lab personnel need to be trained in the methods because clearly a great deal depends upon a reliable test.

Q: Why does the CDC recommend against face masks when the science clearly shows they are exceedingly beneficial at stopping the transmission of flu-like disease? Here’s a study I found on the CDC’s own site clearly demonstrating the efficacy of properly fitted N95 face masks during the SARS outbreak.

A: I did not mean to imply that CDC advises against using face masks. My statement was based on the fact that on the CDC’s swine flu page, there are no recommendations for face masks. This is the page that most of the public will see. Face masks will work if used properly; if not used according to directions, they will not prevent transmission or infection and will convey a false sense of security.

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