Was flu work done 'under the radar'?

One point that Yoshihiro Kawaoka touched uponduring the CEIRS network webinar that I failed to discuss is the idea that the H5 transmission research was done in secret and blindsided the powers that be as has been speculated on in the media.  Let's take a closer look.  

Is that Fouchier or Kawaoka?  See what I did there

  In 2009 The WHO suggested that researchers investigate what it would take to create mammalian adapted H5.  A similar proposal had been issued previously by the NIH in 2006. On top of that, the RO1 grant that paid for Kawaoka's research had 5 specific aims.  The first two specifically proposed the research presented in the controversial paper.  Aim 1) adapt H5N1 from birds to humans. Aim2). induce respiratory transmission.  The methods that his team used to create these viruses have been used previously with other avian influenza viruses to produce similar results (Wan et al., Sorrell et al.).  Finally, all studies involving animal test subjects are reviewed by a panel of university officials, researchers, and outside community members for ethics and welfare. Additionally, the research was reviewed annually by the UW-Madison IBC to ensure no inappropriate research is conducted with potentially dangerous pathogens.

This is not how science happens.

This research was vetted at the local, national, and international level. The studies were not conducted by rogue scientists by misappropriating grant money.  This is not a case of intentionally breaking rules and hoping an apology afterwards is will prevent punishment.  They were not hiding unethical research they wanted to do, but knew others wouldn't understand. ------------>
The university annually inspected it, the NIH approved it and funded it, and the WHO called for it to be done.  To say now that these scientist acted irresponsibly in secret is either revisionist history, or spouting off on something with no understanding of how it works.  But the media would never do that, right?

Where should flu work happen?

With the H5 controversy of the last few months, there has been some talk about moving the mammalian transmissible or even all highly pathogenic flu viruses into BSL4 laboratories.  Here is why it’s a terrible idea.

BSL3, BSL3+, BSL4: What’s the difference?

 

BL3 personal protection

BSL3 is a lab that has certain safety and containment features that go above and beyond a typical molecular biology or chemistry lab (typically BSL2).  BSL3 labs are required to be behind double locked doors. The lab space is required to be maintained under a negative air pressure so that opening doors or unintentional breach of containment causes air to be pulled into the lab as opposed to it  (and what ever is in it) being blown out of the lab.  Additionally, all waste generated is required to be recovered and decontaminated prior to disposal.  Finally, all personnel working in a BSL3 lab are required to wear additional personal protective equipment (PPE). This includes disposable lab coats, multiple disposable shoe covers and multiple gloves, as well as surgical style facemask.

Bl3+ personal protection

BSL3+, other wise known as BSL3 agriculture or BSL3 enhanced, has significant upgrades to the security and safety measures of BSL3.  A BSL3+ is required to have a pass through autoclave so that all solid waste is decontaminated prior to leaving the facility. The researchers body is the only thing that leaves BSL3+ without going through the autoclave (with very few exceptions and only with proper permission and safety protocols).  The scrubs they wear under their PPE are removed in the facility and the researchers are required to shower out in order to leave. PPE is similar to BSL3 but also include a Tyvek coverall and a loose fitting overhead PAPR (powered air purifying respirator) that is equipped with a HEPA filter to scrub the air they breathe. The facility is sealed airtight and all rooms are monitored for pressure changes multiple times a day and the air itself is double HEPA filtered. The entire facility can be decontaminated via gas decon ports from the outside. All security features have built in redundancies and back ups.  Prior to gaining access to a BSL3+ lab, a worker must undergo extensive training and testing and must pass an FBI background check.

A BSL4 lab is the highest level of biosafety and containment possible.  It includes all the elements of a BSL3+ lab with two main differences. Researchers are required to wear a fully sealed suit that air is pumped into from either the outside or a sealed air tank.  These are the moon suits you see in movies like ‘Contagion’ or ‘Outbreak’.  Additionally, the researchers take a chemical decontamination shower to exit the lab. 

BL4 personal protection

Where should it be? 

 

Currently all work done in the US or with US grant money utilizing highly pathogenic influenza is performed under BSL3+ conditions.  All facilities are inspected annually by relevant agencies depending on the pathogens in use. This can include the CDC, NIH, USDA and others. Are these safety features enough for these viruses? BSL3 and BSL4 are reserved for work with pathogens that can cause severe to fatal infections in humans.  The difference between BSL3 and BSL4 is that BSL3 agents have known and effective treatment strategies and BSL4 agents do not.  The only exception being smallpox in BSL4 due to the fact that it has been eliminated from nature.  With this in mind, where should mammalian transmissible H5N1 or all highly pathogenic influenza strains be?  There is no doubt that highly pathogenic influenza is a very deadly pathogen. The ability to become airborne means standard BSL3 conditions are probably not adequate protection.  However the use of PAPRs in BSL3+ are capable of filtering virus out of the air.  The research done with these viruses is very clear.  The drugs and vaccines we currently have are still very effective against these strains making BSL4 inappropriate.  Finally, highly pathogenic influenza is found readily in nature. Humans are exposed to it daily.  All of this leads to one very clear conclusion:  Highly pathogenic avian influenza, mammalian transmissible or not, does not belong in BSL4.  BSL3+ is sufficient protection.  A move to BSL4 would be unnecessarily disastrous, not only to influenza research, but also all current BSL4 pathogens.  A move to BSL4 would kill many flu labs do to a lack of appropriate facilities. Those flu researchers who would still have access to BSL4 facilities would then be jockeying for time and space inside the facilities that already house other research groups.  Retarding research on understanding the most dangerous pathogens is an indescribably terrible idea.  

Even Charles Barkley knows the move is a bad idea.

More on Kawaoka's H5

Kawaoka presented his H5N1 study to the CEIRS network of researchers today via webinar.  Most of this has been covered in other write-ups describing his presentation at the Royal Society earlier in the month, but I will expound on some things that I feel have not been covered in what I have read.  I have a more detailed description of the experiments and results as well as some background on flu here. 

The experiment:

The researchers created an H5 HA that had greatly increased human cell specificity due to three mutations that were randomly added: N158D, N224K and Q226L. They put this HA with the other genes form the 2009 pandemic H1N1 and this virus was able to infect ferrets and showed very weak aerosol transmission (2 of 6 ferrets from 5 to 7 days after infection).  They took a sample of the virus from the animal that was infected via aerosol transmission and re-infected another ferret.  This time the virus transmitted more efficiently (4 of 6 ferrets, 3 to 7 day after infection).   When they sequenced this virus they found one additional mutation, T318I.

They also tested various combinations of the four mutations to dissect how they affect the behavior of the virus. The first three mutations are at positions that are known to affect the species specificity of a virus. The mutations at 224 and 226 affect the shape of the part of the protein that binds to cells. One set of amino acids cause the virus to bind bird cells, the other causes it to bind human cells, kind of like different keys fit in different locks. The mutation at 158 is known to eliminate a glycosylation site. Of all the mutations outlined in the study, this is the only one that has been found in nature in H5 viruses. Many if not most of H5 viruses isolated from birds since 2009 have lost this glycosylation site and every single human H5 lacks it.  According to Dr. Kawaoka’s work, this mutation also serves to stabilize the HA protein. The final mutation also serves this same function.

To me, the importance of this work is not that this is likely going to arise in nature and we should fear it.  The potential of this virus to occur naturally is very low since there would have to be reassortment followed by passage in the right host to build these or similar mutations. The real take away from this paper is how the mutations interact to create a transmissible virus. The two mutations in the receptor binding domain of HA (224, 226) play a major role in switching the host range of the virus, but it also causes the HA to become unstable. This instability prevents this virus from being very infectious and blocks transmission. The mutation at 158 further pushes the virus towards human cell preference but also increases the stability of the protein.  This allows the virus to be more infectious in ferrets and also gives it the ability to transmit through the air (albeit very poorly).  The final mutation at 318 greatly increases the stability of virus and allows the virus to be more infectious and to transmit more efficiently, though still far less infectious and efficient than the 2009 pandemic virus.

With more research on how various mutations affect the performance of a virus, we can start to build an index of mutations and functions.  When surveillance teams isolate new H5 viruses they can identify how new viruses differ from older viruses and can quickly identify how these mutations affect the virus. If officials can identify virus population before they become airborne then vaccine and drug stockpiles can be used more efficiently.  Rather than fully mobilizing our response efforts every time the virus pops up, we can take a more “don’t fire til use see the whites of their eyes” approach and save our ammunition to utilize in the most effective manner. This will ease strains on what experts believe are inadequate stockpiles of drugs and vaccines.

Vs.

Finally we hear about Kawaoka's paper

Last week the NSABB announced that the two controversial papers discussing mammalian transmissible H5N1 authored by Yoshihiro Kawaoka of University of Wisconsin Madison and Ron Fouchier of Erasmus Medical Center.  The bare minimal story in his paper was revealed by Ron back in September at a conference in Malta and again in February at a meeting of the ASM. However, nothing had been revealed about Kawaoka's work until yesterday.

                                       Kawaoka and Fouchier.  credit: M. Enserink
                                                        From: Sciencemag.org
  
Long story short, I am even more confused as to why this work was blacked out in the first place and a little underwhelmed by the results. I guess that is why the board voted unanimously to allow his work to be published.  Basically, Kawaoka took the HA (the protein responsible binding and entering cells; also responsible for species specificity) from H5N1 made a bunch of variations with a bunch of mutations and checked each one for human specificity. He then took a human specific version and plugged it into a virus with other genes from the 2009 pandemic virus.

To recap, this is a virus with 7 fully human flu genes, plus an "avian" gene that has been heavily modified to look human.  When they analyzed the HA gene they found 4 mutations. N158, Q226L, N224K, and T318I.  If you think of a protein as a chain, Q226L means the 226th link in the chain was changed from a Q, glutamine, to L, leucine.  Three of these mutations are fairly well understood.  N158 sits right near a glycosylation site.  Glycosylation sites are where sugar molecules are stuck to the protein and can be thought of kind of like a costume for the virus. Changing the pattern of sugar on the proteins can alter where the virus can bind and how the immune system "sees" and responds to the virus.  The loss of glycosylation right near this mutation has been known to cause a preference switch to human cells.  The mutations at 224 and 226 sit directly in the area that actually binds to the cell surface, the receptor binding site, or RBS.  Making these mutations physically changes the shape of the RBS so different cellular parts fit it. Its like those kid toys with the shaped blocks that fit in different holes. With one set of mutations you have a triangle hole that fits the triangle pegs on a birds cell. Change those to another set and you get a circular hole that won't fit bird pegs anymore, but will fit the human circular pegs.** The last mutation, T318I,  I have no idea about.  It will be interesting to see the follow up experiments they do with that mutation.

                                                  Child's toy or deadly flu mutations?

The virus Kawaoka's team made was able to infect and transmit by respiratory droplets.  Basically when a ferret sneezes it looks like this:

That cloud of white mist is chocked full of virus if the ferret is infected and with the right set of proteins and mutations may be able to infect ferrets (or people) from several feet away. Ferrets that were only in contact with the virus through they air they shared with infected ferrets, but the important thing is that none of the ferrets died. It also seems that these ferrets didn't get as sick as ferrets that had been infected with seasonal or 2009 pandemic viruses.

So we have a highly unnatural, reassorted virus that causes limited disease, but does transmit through the air.  What the hell was the last 4 months about?


**slightly more complicated than triangles and circles.

New dual use federal policy announced

The federal government revised its policy last week regarding potential dual use research. Dual use research is essentially any research that could be used for both good and evil purposes. The research to initiate the new policy was done on highly pathogenic H5N1. Scientists discovered mutations that would allow these deadly viruses to transmit in mammals. This gives researchers, surveillance workers and public health officials a better understanding of what to look for and how these viruses work. However, fears arose that this information could be used by terrorist to make a bio-weapon; thus the dual use designation.

The policy identifies 15 high risk pathogens and toxins and has called for all funding agencies to identify all federally funded research involving these agents within 60 days. Within 90 days the agencies are required to report all instances of research involving the 15 agents that could be considered dual use. Furthermore, the funding agency, institution and lead scientists of studies found to have dual use risks are to create a "risk mitigation plan". This could include modification of methodology, moving research to more secure labs, and alteration of how the research is communicated to the public and scientific community.

The funding agencies "will determine whether it is appropriate to:
request voluntary redaction of the research publications or communications, classify the research, not provide or terminate research funding."

Previously, research done at the NIH and the CDC was reviewed for possible dual use. This was done only for in house studies. The new policy would apply this same standard to all research carried out with NIH or CDC funding. Here is the kicker: these reviews are to be performed for <i>all current<i/> studies as well as future studies. It also appears this will affect research funded by other government agencies such as the USDA and the DOD.

I get why this policy was made. I agree with the vast majority of it. I am not sure how I feel about grandfathering in current research. It doesn't seem fair to drastically change the game half way through.

"Oh you used the CDC grant to conduct your thesis research on HPAI H7N1 and you are about present your 5 years of work to your PhD committee. Sorry, we just classified that so you can't tell anyone about it. Good luck getting a post-doc though."


The 15 agents are:
a) Avian influenza virus (highly pathogenic)
b) Bacillus anthracis
c) Botulinum neurotoxin
d) Burkholderia mallei
e) Burkholderia pseudomallei
f) Ebola virus
g) Foot-and-mouth disease virus
h) Francisella tularensis
i) Marburg virus
j) Reconstructed 1918 Influenza virus
k) Rinderpest virus
l) Toxin-producing strains of Clostridium botulinum
m) Variola major virus
n) Variola minor virus
o) Yersinia pestis

The 7 types of dual use research:
a) Enhances the harmful consequences of the agent or toxin;
b) Disrupts immunity or the effectiveness of an immunization against the agent or toxin without
clinical or agricultural justification;
c) Confers to the agent or toxin resistance to clinically or agriculturally useful prophylactic or
therapeutic interventions against that agent or toxin or facilitates their ability to evade
detection methodologies;
d) Increases the stability, transmissibility, or the ability to disseminate the agent or toxin;
e) Alters the host range or tropism of the agent or toxin;
f) Enhances the susceptibility of a host population to the agent or toxin; or
g) Generates or reconstitutes an eradicated or extinct agent or toxin listed in Section

Good news all around

The NSABB has decided to allow the two transmissible H5N1papers to be published in full after new versions of the papers were reviewed. Very little is known about the Kawaoka experiments. However, the limited information on the Fouchier experiments seem to point to a virus that is lethal when large doses are applied directly to the lungs, but non-lethal when infection is initiated by “natural” aerosol transmission. Typically this route of infection leads to smaller doses that end up in the upper respiratory tract(nose, trachea) as opposed to the lower respiratory tract (lungs). WARNING: pure speculation ahead ~*I am guessing that the revised version of the paper will clearly point this out and will include data from experiments carried out since the original submission of the findings. As far as dual use is concerned, its would be prohibitively hard for terrorists to get large doses of a virus into target humans lungs and even then there would be no lethal transmission. Makes for a pretty bad bioweapon.*~

The board voted unanimously to allow for publication of the Kawaoka paper with a 12-6 decision on the Fouchier paper. Comments made by some members of the board several weeks ago suggested that the issue the board had was with the transmissibility of the virus, not necessarily the lethality.

“The central issue for me is the transmissibility. Unless Ron [Fouchier] gets up there and says this is no longer mammalian transmissible," NSABB member Arturo Casadevall [would not change his mind].

“The issue is you have a virus generated in laboratory that's now transmissible [in mammals],” says Casadevall, “This virus has the capacity to recombine, and we have no idea what will come out.”

“They not only changed the host range of a dangerous pathogen, they also changed its mode of transmission… All the other differences in methods, or new or clarified work on virulence in ferrets, does nothing to change those facts." Lynn Enquist.

Despite those two comments, it appears (at least in the Kawaoka case) the board did in fact change their minds about a transmissible H5. I am very curious about the differences between the two papers that led to the different voting records. Also, its unfortunate that we will never get to see what was revised in the manuscripts. It would be interesting to see what the board was thinking and what led to the reversal of position.

http://news.sciencemag.org/scienceinsider/2012/03/breaking-news-nsabb-reverses-pos.html?rss=1

Anti retrovirals help malaria drugs

Researchers revealed at the 19th Conference on Retroviruses and Opportunistic Infections clinical data showing anti-retroviral drugs coupled with standard anti-malaria treatment greatly improves the efficacy of the malaria treatment. The study was done by a team of researchers headed by Diane Havlir of the University of California, San Francisco (UCSF), and Moses Kamya of Makerere University College of Health Sciences in Uganda

The data, presented by Jane Achan, showed that treatment with protease inhibitors lopinavir and ritonavir (two anti-retrovirals) resulted in a 41% drop in malaria cases. Breaking the data down even further revealed a 29% reduction in first malaria cases, or cases in which the children had no previous malaria infection. When they isolated reinfections they found a 59% reduction. These drugs are mildly protective against an initial infection, but are very good at blocking a secondary infection. Why is that?

The common anti-malaria drug, Lumefantrine, can stay in the body for several weeks, extending the length of time the drug protects. Ritonavir is a protease inhibitor that has little effect on HIV, but instead prevents the body from breaking down other HIV drugs and improving the effectiveness of the treatment. It appears that it provides the same effect for anti-malaria drugs as it does for anti-retroviral drugs. When they looked in the blood of children in the treatment groups they found fivefold more lumefantrine in the group that also received the protease inhibitors.

"We think that these higher lumefantrine exposures were really what was driving the protection against recurrent episodes of malaria," Achan said.

It appears that once again, the HIV world has turned science dogma on its head.

"We always think of drug-drug interactions as something to be avoided, but HIV has taught us the opposite," says Paul Volberding, an HIV/AIDS clinician at UCSF.

These findings could alter how treatment occurs in areas with high incidence of both HIV and malaria. Additionally, the study could change how long term prophylactic malaria treatment is done.

http://news.sciencemag.org/sciencenow/2012/03/hiv-treatment-fends-off-malaria.html?rss=1

UPDATE: H5 controversy

http://www.sciencemag.org/content/335/6073/1155.full?rss=1

Don't know how many people can read this, but I will overview the highlights.

Fouchier inoculated 4 ferrets with a high dose of the mutated H5N1 intertracheally. These ferrets were able to transmit via aerosol to 3 of 4 contact ferrets.
He then took samples from one of the 3 infected aerosol contact ferrets and infected two more ferrets. The virus was then able to transmit to 2 of 2 ferrets via aerosol. In total that is 5/6 for aerosol transmission. 0 of 6 aerosol infected ferrets died. 6 of 6 directly inoculated ferrets died.

Recap: large amounts of virus to the lungs kills ferrets and allows the virus to spread via aerosol transmission. Ferrets that are infected via aerosol shed less virus and do not die. When the virus is directly inoculated it kills ferrets at a similar rate as the wild type H5N1. When ferrets are "naturally" infected via aerosol contact, infection is much less severe. "Doomsday virus"? Hardly NYT. Sensationalize your story that has no real information backing it up much? Much too much. Sadly this is what the American and international media has become.

So the "report by ABC News on 18 February that said aerosol transmission killed 'all 40 of the exposed animals' in neighboring cages" is pretty seriously wrong. Ron's work was pretty seriously misrepresented. He is now catching flak for misrepresenting data (even thought he didn't). What are the chances ABC News is ever held responsible for grossly misleading the public? Don't hold your breath.

Additionally they did follow up experiments that were not in the paper. They gave lower doses of the virus internasally. "Only one of eight ferrets infected with the mutant virus through nasal swabs developed severe disease, and none died."

"There are a lot of misperceptions about what you can and cannot conclude from these studies,” Fouchier said at a panel discussion on the topic.

“'This was overwhelmingly less about new data than making sure there was a clear understanding of the old data,' Fauci told Science" in regards to the recent discussion Fouchier had at the ASM.

“'The issue is you have a virus generated in laboratory that's now transmissible [in mammals],' says NSABB member Arturo Casadevall, a microbiologist at the Albert Einstein College of Medicine in New York City. 'This virus has the capacity to recombine, and we have no idea what will come out.'" So the prudent thing to do is lock the virus and the information up tight so virtually no work can be done on it? Very short sighted.

I am just glad Fauci seems to have come around.

A rant on a personal pet peeve

I was just forwarded an article from weather.com about the delayed flu season. It was one of the worst science articles I have ever read. The article clearly demonstrates why science education is necessary for basic everyday living and how this country is grossly scientifically illiterate. The premise of the article is that the delayed flu season is "bizarre" and will now coincide with allergy season. The author shows some surveillance maps from the CDC to back her claim and relayed a story about a school district in Missouri that had to close because of the "flu". She then quotes the superintendent who talks about norovirus (wait what), then a Dr. about the 'stomach flu' (uncontrollable rage building). The rest of the article she spends interchanging facts about norovirus and influenza as if they are the same thing including a link on where to get the flu vaccine followed by tips on how to avoid the flu, that are actually tips on how to avoid noroviruses. (FULL ON RAGE-HATE).
...
(Deep breath Brian. Calm yourself.)
...
First things first. There is no such thing as 'the stomach flu', 'the 24 hour flu', or 'a touch of the flu'. Flu is caused by the influenza virus and only the influenza virus. It is a respiratory disease; it affects your lungs, your airways, and your nasal passages. If you get the flu you are not touched and it does not last one day. You are hit full force with a sledgehammer of sick for three to seven days.
Norovirus on the other hand is an enteric virus (stomach bug). It symptoms include feeling like a bomb went off inside of you, moderate to severe diarrhea, and vomiting (age dependent) for about 24 hours. Noroviruses transmit via fecal-oral contamination. Someone doesn't wash their hands after "a bathroom episode" and uses a doorknob/handrail/atm/etc. You then use that doorknob/handrail/atm/etc. and then pop a stick of gum in your mouth/adjust your lipstick/absentmindedly chew your nails and BAM! you're next. Flu can spread in a similar manner (minus the fecal part), but also is transmitted through the air.
"Oh Brian," you might say, "you're just arguing semantics. What does it matter if people call it 'the stomach flu' or not?" It matters because of perception. When you have a tummy ache and your Dr. says "you have a touch of the flu or the 24 hour flu." You go home and eat some chicken noodle soup and the next day you feel better and head to work. Next year rolls around and its time to get the flu shot. You think to yourself, "Shots sucks. I had the flu last year and it wasn't so bad. Uuuhhhh... Yeah, I'm gonna skip it." Vaccination rates drop. Then just to be safe you decide to head to the internet to get some tips on how to avoid the flu. You end up on a very heavily traveled site and read an article that freely interchanges three or four different pathogens and diseases and you make a note of the table titled "Ways to keep the flu at bay" that actually contains a list of how to prevent norovirus. You are now unvaccinated, religiously cleaning your toilet, and disinfecting your kitchen. You are unprotected against the flu, but hey, at least you probably won't end up with the screaming squirts.

Ok. Rant over. Here is the link if you want to get dumber. Please don't share this with anyone else. I have written a strongly worded letter to weather.com expressing my dismay at this article and really hope others do too (when did I turn into a cranky old man?).

http://www.weather.com/outlook/weather-news/news/articles/stomach-flu-outbreak_2012-03-06?page=1

Dan Perez and Andrew Fauci debate the H5 controversy.

http://link.brightcove.com/services/player/bcpid46211837001?bckey=AQ~~,AAAABvaL9Hk~,mLC66bU8hPPvh8_ouk-nThborTB9hP1y&bclid=0&bctid=1447446742001

Flu researchers volunteer to temporarily stop research

So my boss, amongst other prominent flu biologists have 'volunteered' to not do H5 transmission work for the next 60 days and to form an international forum to discuss the implications of this and similar research. This forum will include researchers, public heath officials, and governmental representatives and will hopefully result in some more level headed decisions being made on this subject, and in the future. No word yet on whether or not the chickens and swine are joining the moratorium on creating dangerous viruses.

:http://www.sciencemag.org/site/feature/data/hottopics/biosecurity/Fouchier.Express.pdf