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Flu Science for Dummies

by: SusanC

Tue Jan 09, 2007 at 13:19:39 PM EST


( - promoted by SusanC)

We used to have a Dummies' corner thread in the old forum
SusanC :: Flu Science for Dummies
which was favorites with a lot of people.  Some very good questions were asked, and some important insights were gained.  I thought it would be good to start this up again, but use separate diaries for science, preps, html etc.  Since there are a few prep and html Q&A type diaries already, I will put this one up for science questions, like virology, vaccines, 1918, whatever...

I would suggest we treat this as another community thread, so anyone can ask questions, and anyone can respond!

I'm going to cheat a little and start by re-posting part of a comment from the Egypt thread.  ;-) 

Q: I understand that the H1N1 from 1918 must be different from the present H1N1 flu - despite the name share - but how would you explain why and how different in terms that would not require the presence of a glossary?

A: The difference between the 1918 H1N1 and the current H1N1 is that it has had almost 90 years of human to human transmission.  The 1918 virus was a very avian-like virus, ie it was more adapted to birds and not very adapted to human beings.  (It was more adapted to humans than whatever ancestor virus it came from, enough to cause the 1918 pandemic.)  As it worked its way through the human hosts, it came under the attack of human immune systems.  In response, the virus keeps changing its molecular structure so that it will continue to survive, and the H1N1 has done very well in that.  The longer it spent in humans, the more different it became from the original.  However, it is still a descendant of the original 1918 H1N1.

Now, H5N1, even though it is an N1, did not arise from the 1918 H1N1.  Current science shows that it probably had a common ancestor with the 1918 virus, going even further back from 1918.  But because it had not spent almost a century adapting to humans, but has, as far as we know, evolved in birds over all this time, it is a very different N1 virus, and is still more adapted to birds than humans.

The rest of the comment is here http://www.newfluwik...

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Tamiflu resistance in Egypt?
Do you know of any other study/opinion on Tamiflu resistance?

I think it was late administering or inadequate dosing of Tamiflu that preceded the death of the 15F in December 2006 in Egypt. Is 75 mg per day a prophylactic dose i.e. not a treatment dose?  If a prophylactic dose is administered (too late) to an infected patient, would it cause resistance to be built for the strain if subsequently transmitted?

Niman thinks this is evidence of Tamiflu resistance caused by recombination preceding the infection of the 15F.

http://pfiforum.com/...

--------------------------------------------------------------------------------------

niman  Posted: Mon Jan 08, 2007 2:04 am  (page 8 of all posts) 

The 15F was placed on Tamiflu within 24h of developing symptoms. She died a few days later. All four confirmed cases in Egypt this season have died.

--------------------------------------------------------------------------------------

niman  Posted: Mon Jan 08, 2007 3:13 am  (page 9 of all posts)
The NA sequence would show Tamiflu resistance.

I believe she was taking 75 mg per day.

--------------------------------------------------------------------------- 

You want perspective. I want perspective. Let's talk. We don't have to agree on every thing. If we do, one of us is redundant.


tamiflu resistance
I looked at the comments, and my first thoughts are we really are not sure that she got tamiflu within 24 hours of onset of symptoms.  And the 75mg seems to be from Niman only, or at least I didn't see any source quoted.

But assuming that is all true, I have several thoughts.

1. tamiflu is not 100% effective, even in seasonal flu, so you would expect some treatment failures.

2. treatment failure is not the same as resistance.  For example, De Jong http://content.nejm.... published a case in Vietnam where they gave tamiflu quite late, but they were able to document a very fall in virus titre, but the patient still died. (patient 3 in diagram)  That means the virus was sensitive to tamiflu, but the disease process had progressed beyond the point where a reversal of viral titre was enough to reverse the disease.  This is a particularly marked feature with H5N1 infections, and it is thought that the hyper-induction of immune reactions (ie the so-called 'cytokine storm') sets up a cascade that becomes unstoppable after a certain point.

3. to demonstrate resistance, you have to demonstrate a failure in reduction of viral load (patients 1 and 4 above), ideally also showing sufficient drug level (eg not due to failure of absorption of the drug) and/or by specific laboratory tests showing failure to inhibit the growth of virus in some way.  ie NA inhibition assays.

4. drug resistance is not primarily diagnosed by sequence data.  You document whether there is resistance, as above, then you can look at the sequence to see if there are mutations that might suggest the source of this resistance.  There are some specific mutations associated with tamiflu resistance, eg H274Y, which was present in both patients 1 and 4 above, but a virus can also be resistant and not show any identifiable mutations, as shown in this survey of >2000 isolates by Monto et al http://aac.asm.org/c... 

5. assuming the virus does turn out to be tamiflu resistant, the next thing I would like to know is is the sample taken before or after starting treatment?  Are there other samples or mixed isolates?  What about samples from others in the same cluster?  In De Jong's study above, he demonstrated this very dramatically by isolating both the mutant ie H274Y and the wild-type 274H from patient 1, as well as isolating only wild-type 274H from patient 2, who was patient 1's mother, from whom presumably she caught the virus.  So here you have a clearly documented trail of resistance following possibly inadequate dose treatment with tamiflu. 

6.  Tamiflu resistance due to inadequate dose is a known problem particularly in children, as their metabolism of the drug is such that a higher dose per body weight needs to be given when compared to adults.  In this instance, 75mg for a 15-year-old is definitely inadequate, IMO.  However, several studies such as this one http://www.journals.... also show, especially with this H274Y mutation, that it is associated with reduction in receptor binding, so that it is less viable as a transmissible agent. 

7. What would be most worrying is the appearance of naturally occurring or wild-type tamiflu resistant viruses or with H274Y.  So far, eg from the above Monto study, that appears to be distinctly rare, and they all appear to be less 'fit' for transmission. 



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
See Egypt III diary latest news from Theresa42
to which I replied:

http://www.newfluwik...

Simply too little too late with Tamiflu.  I won't call that proof of Tamiflu resistance.

You want perspective. I want perspective. Let's talk. We don't have to agree on every thing. If we do, one of us is redundant.


[ Parent ]
OK thanks n/t




All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
more on tamiflu efficacy
just to put several references in one place.

  1. Animal study, Monto Virulence May Determine the Necessary Duration and Dosage of Oseltamivir Treatment for Highly Pathogenic A/Vietnam/1203/04 Influenza Virus in Mice

  2. De Jong in Vietnam Oseltamivir Resistance during Treatment of Influenza A (H5N1) Infection

    Note that in both studies, but especially de Jong, the title and the main drift of the study appears to focus on resistance.  But when you read it carefully, you realize they are saying that it works.  So be careful if you are going to quote them.

  3. Another paper by de Jong Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia shows the correlation between high viral load and fatal outcome.  Which means that reduction of viral load would be the goal of treatment.  And both #1 and #2 show that tamiflu reduces viral load.

  4. Here's a paper on tamiflu resistance based on surveillance of seasonal flu in the first 3 years of use Detection of Influenza Viruses Resistant to Neuraminidase Inhibitors in Global Surveillance during the First 3 Years of Their Use

  5. WHO Rapid Advice Guidelines on pharmacological management of humans infected with avian influenza A (H5N1) virus 
    This one is useful in specific contexts, eg if your local officials want 'official recommendation before buying tamiflu.

    Recommendations for treatment of patients with confirmed or strongly suspected human infection with the H5N1 virus

    Where neuraminidase inhibitors are available :

    • Clinicians should administer oseltamivir treatment (strong recommendation); zanamivir might be used as an alternative (weak recommendation). The quality of evidence if considered on a continuum is lower for the use of zanamivir compared to oseltamivir.
    • Clinicians should not administer amantadine or rimantadine alone as a first-line treatment (strong recommendation).
    • Clinicians might administer a combination of a neuraminidase inhibitor and an M2 inhibitor if local surveillance data show that the H5N1 virus is known or likely to be susceptible (weak recommendation), but this should only be done in the context of prospective data collection.





      All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
It is my understanding that
patients are tested first before any treatment is administered. I'm sure they didn't just "give" this person because they showed up sick. Doctors, to my understanding, asess, dignose,and prescribe treatment. And this can take a few hours, when lab tests are involved? Am I wrong in this?
It would seem to me, that they took the sample first, before even giving Tamiflu.

United we stand: Divided we fall
www.flunewsnetwork.com


[ Parent ]
Please check these links
http://www.newfluwik...

http://www.who.int/c...

QUOTE

Both patients had been on treatment with oseltamivir for two days before the clinical samples that yielded the viruses were taken.

END QUOTE

http://www.newfluwik...

QUOTE

They decide to cut down poultries at the time of the weddings of Chaféya which take place on December 14.  The evening, Intissar is the first to be suffered from an excessively high temperature "We do not have a medical unit in our village.  Nearest is in the town of Zefta, to 12 km from here.  Thus we tried to treat Intissar with residence, but without success.  Its [her] health started to worsen so that it did not manage any more to breathe.  The following day, Réda and Chaféya fell sick too.  Three days after, we hospitalized them all in Zefta where they received during one week a treatment against the normal fever...," remembers Abdel-halim, the father of Intissar and Réda, old of 70. 

END QUOTE

By the time they administered Tamiflu (way pass 48 hours, and of course pass 24 hours deadline for the young), and only a prophylactic dose of 75 mg per day (per Niman), the virus would already have been at a very advanced stage in the body.  Then two days later, samples were taken that yielded the Tamiflu moderate resistance test result.

You want perspective. I want perspective. Let's talk. We don't have to agree on every thing. If we do, one of us is redundant.


[ Parent ]
in a perfect world, yes
but we know it's not perfect, so no, that doesn't happen.

It would seem to me, that they took the sample first, before even giving Tamiflu.

This is official, from the WHO:

The two patients from whom samples were taken were a 16 year-old female and a 26 year-old male from Gharbiyah Province, Egypt.  They were a niece and uncle, respectively, who lived in the same house. The girl was admitted to a hospital on 19 December 2006, while the man was admitted on 17 December. On 21 December they began receiving 2 tablets per day of oseltamivir. On 23 December they were moved to a referral hospital. The samples which have so far been tested were taken from the two patients on 23 December. The girl died on 25 December and the man died on 28 December 2006.

Plus, the major problem IMO is not in the testing, it's in giving the meds early enough.  I'm willing to not be 100% sure if these or other patients indeed had H5N1 if we can treat them early enough to save their lives.  We can always use serology to confirm later. 

The guy got tamiflu 4 days after admission, the girl, 2 days.  Think about it....

It took them 4 days to consider avian flu as a possible diagnosis.  What makes governments like the UK believe that people can get tamiflu within 12 hours of onset of symptoms in the pandemonium of an unfolding pandemic?



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
Ditto.
What makes the CT Public Health Dept. think that they can get Tamiflu to the kids in my town within 21 hours of onset of symptom too? 

Will they just give out this (by then very valuable and scarce) medication with out first testing?  How long will it take to get test results back? 

If they decide to give Tamiflu anyway before testing, can the guys in charge actually get it to the doorstep of who needs it in 12 hours?  In the chaos of a pandemic?  And talk about worried well.  Every mother in town will call the DPH if her child looks pale or begins to run a fever. 

The utility is SO narrowly defined for Tamiflu - that 12 hour window, particularly for children - that physicians really cannot, medically, justify not prescribing this medication in advance (only enough for one family) so that it can be used, and used properly, when it is needed. 

And, my state Pandemic Plan clearly states that they want ill people to be treated at home anyway.  They should, then, tell the people that, and give them the chance to obtain the proper tools to do so - i.e. masks, gloves, fever reducers, and antivirals, particularly Tamiflu because of its unusually narrow requirements.


[ Parent ]
;-) same for the UK
yeah, I kinda scratched my head over the 21...



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
typo fingers :-)


[ Parent ]
and hey,
at least you guys will stand in line better than we will for the Tami, etc.  Or should I say "queue up."

[ Parent ]
In the case of the Egyptian patients, I remember they finally admitted
having contact with poultry, after doctors realized that the symptoms were too much bird flu like, then transferred and given the prophylactic dose of 75mg.  Because it was obviously quite late from symptom onset, I don't think the authorities would have waited longer to get tests done before administering Tamiflu.

You want perspective. I want perspective. Let's talk. We don't have to agree on every thing. If we do, one of us is redundant.

[ Parent ]
resistance in one individual vs in circulating virus
This is re-posted from here in answer to the following question:

I have a medical question

In all the years of medical history, has it ever been found that a virus has became drug resistant so quickly? It has taken years for many of our antibotics to become ineffictive on many viruses. This virus seems to have become resistant over a period of a year? Tamiflu, to my knowledge hasn't been used in treating this for that long in humans cases, has it?
by: cottontop @ Thu Jan 18, 2007 at 09:16:03 AM EST

Drug resistance can develop very rapidly, and we are talking about 2 things.

One is the development in one individual of resistance during treatment.  This can happen quite quickly, since flu viruses replicate rapidly and can mutate with every single replication.

But this resistant strain may or may not be the dominant and/or virulent strain in the patient.  For example, the very first tamiflu resistant H5N1 report was in a girl who fully recovered, and in whom both the wild type (sensitive to tamiflu) and the resistant strain were isolated.  To the extent that she fully recovered, it means that this resistant variant was not particularly pathogenic to the girl, despite its presence.  OTOH, it could also be the dominant and lethal strain, as in the 2 other patients reported by de Jong in Vietnam, where the resistant strain was isolated in high concentration late in the course of disease and when the patients died.

The other is the development of resistance in the circulating virus.  ie the drug resistant strain is able to be transmitted from host to host (whatever the host might be) and can survive the transmissions.  This requires a certain level of 'fitness' in the virus, an ingredient which scientists are very interested in but of which they know very little in specifics.

In the case of human flu viruses, there are some mutations that confer resistance to NA inhibitors but the virus becomes less transmissible.  There are other mutations that confer resistance but still remain transmissible.

Then there is the issue of virulence.  Remember tamiflu is a neuraminidase NA inhibitor.  The virus needs binding to the NA molecule in order to escape the cell in which it has been replicating, and spread inside the host body from cell to cell.  NA inhibitors compete at the same site.  NA inhibitor resistance could happen by the virus adapting its molecular configuration, but this could at the same time make it less efficient with interacting with the NA molecule.

I use the word 'interacting' because the issue of NA binding is a complex one, and 'good' binding to NA is not necessarily good for the virus, because if the binding is too strong, it is not efficiently released to other cells.  There appears to be some complex balance of binding to haemagglutinin (the HA molecule that is needed for entry into cells) and to NA such that an optimum combination is necessary for efficient pathogenesis.  The exact nature of this interaction is still subject to study.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
What is M230I?
This seems to be a very significant development, at least according to Niman.

Does any one know?

Please check the last sentence of this post about Gharbiya in Egypt:

http://pfiforum.com/...

niman  Joined: 05 Dec 2006  Posts: 115

PostPosted: Fri Jan 05, 2007 7:48 pm  Post subject: Re: Yes  Reply with quote

NawtyBits wrote:
niman wrote:
JWB wrote:

homesteader, I was just about to post the same. Thanx to all who are involved in this. Absolutely Amazing!

You will be more amazed when you see the sequences. A/Egypt/14724-NAMRU3/2006 and A/Egypt/14725-NAMRU3/2006 (accession numbers EF200512 and EF200513) will be public on Wednesday, and the fireworks will start.

Fireworks are typically shot off to celebrate something.....why do I have a feeling we won't be celebrating anything....

------------------------------------------------------------

There are three stages of denial

1 it can't be true

2 even if it is true it can't be important

3 we knew it all along

The Gharbiya cluster will move many from stage 2 to stage 3, as far as recombination is concerned.

On the pandemic front, it will be a major wake-up call.

On the sequencing front, the playing field will be reorganized, and random mutations and reassortment will be put in their appropriate place.

Critical mass has been achieved (and it all started with M230I).

You want perspective. I want perspective. Let's talk. We don't have to agree on every thing. If we do, one of us is redundant.


M230I
I did several searches for that, and the only thing that comes up with M230I is with HIV not flu.  Everything else from google is directly or indirectly from Niman, who appears to think this is a new and significant cos (according to him) it is also present in H3, H1 and influenza B.

230 is right next to one of the receptor binding domain for H5. 

Not having any success, I emailed Taubenberger, who, bless him, did a search of all human H5N1 from 2003-6 in GenBank.  Out of 85 sequences, 3 were M, 82 were I.  The 3 M's were from 2005-6. 

Which means that M230I is not anything new with H5N1.

That's the first thing.

Secondly, you can't compare H5 to H3 or H1 in that way.  Different viruses have different receptor binding specificity 'rules'.  For example, 190 and 225 are crucial for H1 but don't work for H5.  For H3, the significant sites are 226 and 228, but these are only 'minimally effective' (JKT) for changing H5.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
Different viruses have different receptor binding specificity 'rules'.
Are you saying for it to be relevant it will have to be something like  M190I in H1, M226I in H3 to be receptor binding specific?  Put it another way, M230I in H3 is not the same as M230I in H5?

Finding M230I in HIV, H3, H1, and flu B does not make it necessarily more transmissible if found in H5N1?



You want perspective. I want perspective. Let's talk. We don't have to agree on every thing. If we do, one of us is redundant.


[ Parent ]
couple of things
  1. M230I in HIV is not the same as M230I in H3 or H1.  It only means on a particular gene, the 230th position carries that particular change.  It's like #23 Main street is not the same as #23 Hope Street.

  2. M to I mutation does not mean the same thing wherever you see it.  Each individual mutation has to be researched in its own context for significance, even if they happen to be in the 'same' location by the numbering system, eg M230I in H1 or H5 would have different significance.

  3. In this instance, it is not even an M to I 'mutation' in the sense that it always used to be 'M' now it's become 'I'.  The 'I' is actually more common than M in position 230, and was present in all isolates of human H5N1 before 2005. 

Which was surprising to me, but there it is.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
Just out online, Nature Medicine
Tropism of avian influenza A (H5N1) in the upper and lower respiratory  tract Nicholls et al

ABSTRACT:
Poor human-to-human transmission of influenza A H5N1 virus has been attributed to the paucity of putative sialic acid alpha2-3 virus receptors in the epithelium of the human upper respiratory tract, and thus to the presumed inability of the virus to replicate efficiently at this site. We now demonstrate that ex vivo cultures of human nasopharyngeal, adenoid and tonsillar tissues can be infected with H5N1 viruses in spite of an apparent lack of these receptors.

COMMENT:
This is an elegant study that raises new questions about what we don't know.  To simplify, I'm going to talk about 2 groups of cells, those lining the upper respiratory tract or nasopharygeal cells, and those lining the lower respiratory tract.  Out of the second group, I'm going to point out 2 cell types to pay attention to, pneumocytes and macrophages.

Let me first give a list of what is known from previous studies before this one:

  1. avian viruses tend to bind to different receptors than human viruses.  I'll use the short notation 2,3 for avian receptors and 2,6 for human receptors.
  2. 2,3 receptors are present on the surface of pneumocytes but not present or very rare on macrophages or nasopharygeal cells.
  3. H5 tends to bind to 2,3 but not 2,6 receptors
  4. H5N1 grows in cultures of cells from the lower respiratory tract, but the cell type was not determined

From the above, some scientists have assumed that it means H5N1 in its current form can't infect upper respiratory cells.  If this were true, it would mean that we don't have to worry about droplets or ingestion as methods of transmission, only aerosols.

So researchers cultured H5N1 virus samples in various cell types from upper and lower respiratory tracts.  They also verified that the H5N1 samples did bind to 2,3 but not 2,6 receptors.

What they found was that they could culture the virus, and indeed there was a rising viral titre (showing viral replication) in nasopharygeal cells from the upper respiratory tract, as well as both pneumocytes and macrophages.

Since, out of these 3 groups, only the pneumoncytes are supposed to have 2,3 receptors, and since viral replication requires entry of the virus into the cell, these results begs the question:

How did the virus get into the nasopharygeal cells and macrophages if the 'right' ie 2,3 receptors were not present?

There are still lots that we don't know. 



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


It doesn't need the 2,3's then, at all.
But why, if that is so, has it not taken off in those large opportunistic family clusters? 

Which strain of the virus did they study here? 


[ Parent ]
not that it doesn't
I think it is possible there are multiple pathways, and/or more than one component is required.  And since we are talking about complex biological mechanisms, any reductionistic approach (ie taking only one component at a time) including this one, may give you incomplete answers.  Yet science cannot be studied without taking apart the components, that's the dilemma.

Plus the sum total of all components studied separately may not add up.  Like 1+2 may not equal 3.  Don't know if I am explaining that clearly.  Maybe tomorrow when I'm more awake, this is 4am here. ;-)



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
No that makes sense
and I've always thought it was a synergystic mechanism we were looking at.  Maybe more than one synergystic mechanism too, to complicate things.  Otherwise, we would have a lot more 1918's between 1918 and now. 

[ Parent ]
Thanks, Susan!
That was really helpful!

Here's more of the pre-publication material from Nature...

Tropism of avian infuenza A (H5N1) in the upper and lower respiratory tract

1/10/07 Nature Publishing Group

J M Nicholls1, M C W Chan2, W Y Chan2, H K Wong2, C Y Cheung2, D L W Kwong3, M P Wong1, W H Chui4, L L M Poon2, S W Tsao5, Y Guan2 & J S M Peiris2

Poor human-to human transmission of influenza AH5N1 virus has been attributed to the paucity of putative sialicacida 2,3 virus receptors in the epithelium of the human upper respiratory tract, and thus to the presumed inability of the virus to replicate efficiently at this site.  We now demonstrate that exvivocultures of human nasopharyngeal, adenoid and tonsillar tissues can be infected with H5N1 viruses in spite of an apparent lack of these receptors.

Exvivocultures of lung epithelium have been used to demonstrate that H5N1 viruses can infect the human lower respiratory tract 1.  These findings were reinforced by the detection of Maackiaamurensisagglutinin (MAA)-2 lectin binding, which identites the sialic acid (SA) a2-3 linkage (the accepted receptor for avian infuenza viruses), and by the demonstration of direct binding of fluoresceinated H5N1 virus to the lower respiratory tract 1,2.  However, evidence for productive virus replication was not provided and the identity of the virus-infected cells in the lung was unknown.  Also, comparable studies in which exvivocultures of the upper respiratory epithelium were infected were not carried out.  The paucity of MAA2 binding in the upper respiratory tract was assumed to indicate an inability of the H5N1 virus to replicate in the upper respiratory tract 1, which has important clinical and epidemiological implications.  For example, if this were the case, H5N1 transmission would be unlikely to occur via large droplets or ingestion, and would probably require transmission by small particle aerosols (o5 mm droplets) in order to reach the lower respiratory epithelium.

Although previous investigations have demonstrated that avian H1N1 (A/Mallard/Alberta/119/98) virus does infect differentiated nasal epithelium cultures invitro 3, the ability of highly pathogenic avian infuenza H5N1 viruses to replicate in exvivonasopharyngeal organ cultures is not known.  We obtained fresh normal nasopharayngeal biopsies from 15 individuals who were being screened for nasopharyngeal carcinoma and from 6 healthy volunteers who gave written informed consent for a study approved by the Hong Kong University and Hospital Authority (Hong Kong West) Institutional Review Board (Supplementary Methods online).  In addition, fragments of tonsillar and adenoidal tissue were obtained from individuals undergoing elective tonsillectomy and adenoidectomy, and written informed consent for the use of this tissue for research was obtained.  We infected the exvivotissues with avian H5N1, human H3N2 and human H1N1 viruses within 3 h of collection (Supplementary Methods).  We carried out parallel experiments on well-characterized primary human nasopharyngeal epithelial cells in vitro.

We found that H3N2 and all H5N1 viruses tested readily infected epithelial tissues of the nasopharynx, adenoid and tonsil (Fig.1a-d).  There was no viral antigen detected in mock-infected tissues or in tissues infected with ultraviolet-inactivated H5N1.  Productive viral infection in the tissue fragments from the nasopharynx was demonstrated by an increase in the viral yield in culture supernatants (Fig.1e).  We also infected nasopharyngeal tissues (Fig.2a) and primary epithelial cells (Fig.2b,c)at 37.1C.  We found increasing viral yield in the nasopharyngeal biopsies (Fig.1e) and in the primary nasopharyngeal cells (Fig.2d).  Experiments repeated at 33.1C also resulted in infection of the exvivocultures and the primary nasopharyngeal cells (Fig.2e-g), and there was increased viral yield in the primary nasopharyngeal cells (Fig.2h).

To confirm and extend previous findings in the lower respiratory tract 1, we carried out parallel experiments using exvivocultures of lung tissue.  We identfied viral antigen in exvivo-infected lung tissues, and dual labeling of these tissues showed that the virus-infected cells were pneumocytes (Fig.1f) and alveolar macrophages (Fig.1g).  Increasing viral yield in culture supernatants provided evidence of productive viral replication (Fig.1h).  Viral antigen was also readily detected in H1N1-infected lung tissues (Supplementary Fig.1 online).

These findings parallel previous observations that H5N1 and H1N1 viruses can replicate in human primary lung epithelial cell cultures and macrophages invitro 4,5 and are in agreement with a previous study showing virus binding in the lung to type 2 pneumocytes and alveolar macrophages 2.

Lectin histochemistry on the exvivobiopsies and archival tissue using Sambucusnigraagglutinin (SNA), MAA1 and MAA2 showed that MAA2 (which recognizes the sialic acid SAa2-3Gal, believed to be the receptor for avian influenza) bound strongly to alveolar pneumocytes but poorly, if at all, to the upper respiratory epithelium and alveolar macrophages (Supplementary Fig.2 online).  SNA binding was similar to that reported previously 1, being present in the ciliated and goblet cells of the upper respiratory tract and not prominent in the alveolar epithelial cells.  Because H5N1 infection occurs only in cells of the upper respiratory tract that have few SAa2-3Gal receptors (as defined by MAA2 lectin histochemistry), we have to consider alternative receptor binding profiles for these viruses.  MAA1 and MAA2 are two isoforms of MAA, both of....

[snipped]

In conclusion, our finding that H5N1 virus infects nasopharyngeal and oropharyngeal epithelia implies that the inefficiency of the avian-to-human or human-to-human transmission of the H5N1 virus may not be explained by the inability of the virus to replicate at these sites.  Virus infection of cells that apparently do not express SAa2-3Gal.1-3GalNAc implies that there may be other binding sites on the epithelium that mediate virus entry.  Furthermore, because human H1N1 and avian H5N1 viruses do not differ in their ability to replicate in the alveolar epithelium, we also conclude that the increased severity of human H5N1 influenza cannot be explained purely on the basis of a differential tropism of H5N1 to the lower respiratory tract.

1Department of Pathology, 2Department of Microbiology and 3Department of Clinical Oncology, University of Hong Kong, Pok Fu Lam Road, Hong Kong SAR, China. 4Department of Cardiothoracic Surgery, Grantham Hospital, Wong Chuk Hang Road, Wong Chuk Hang, Aberdeen, Hong Kong SAR, China. 5Department of Anatomy, University of Hong Kong, Pok Fu Lam Road, Hong Kong SAR, China. Correspondence should be addressed to J.M.N. (nicholls _at_ pathology.hku.hk) or J.S.M.P. (malik _at_ hkucc.hku.hk).

Received 11 July 2006; accepted 4 December 2006; published online 7 January 2007; doi:10.1038/nm1529

Note: Supplementary information is available on the Nature Medicine website.

ACKNOWLEDGMENTS

We thank K. Fung, C.Y. Sing and I.H.Y. Ng for their technical expertise.  This work was supported by the Wellcome Trust UK, by Research Fund for Control of Infectious Disease grants 03040872 and 06060552 from the Health, Welfare and Food Bureau, Government of the Hong Kong Special Administrative Region, People's Republic of China, and by Research Grants Council Central Allocation HKU 1/05C from the Government of the Hong Kong Special Administrative Region, People's Republic of China.

AUTHOR CONTRIBUTIONS

J.M.N. and J.S.M.P. designed and coordinated the experiments.  M.C.W.C., W.Y.C., H.K.W., C.Y.C., L.L.M.P. and Y.G. performed the infection with influenza viruses and interpreted the results.  D.L.W.K. provided the upper respiratory tract biopsies, and W.H.C. provided the lung tissues.  M.P.W. interpreted the results of the lung biopsies, and S.W.T. provided the human primary nasopharyngeal epithelial cells.

COMPETING INTERESTS STATEMENT

The authors declare that they have no competing financial interests.

Published online at http://www.nature.co...

Proud FAF-er.


[ Parent ]
oh yeah
I forgot to include more quotes.

Thanks, Theresa42.  Helpful as always.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
Thank you!
For making it make sense!  :)

Proud FAF-er.

[ Parent ]
btw revere has a good blog
on this paper at Effect Measure

http://scienceblogs....



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
Yes Yeah and All that
So glad to see flu science for dummies back. I read the dairy and learned at least three things. Now to pick your brain. Why arent we seeing more dead mammals in areas like Indonesia, if the virus is in the enviorment ahouldnt we see rodents feral cats [seen a few of those] and other scavenger species dying in large enough numbers to notice?
THANKS :-)
janetn

...
along those lines, if they had mammal impact in Indonesia, would they investigate/report it?

[ Parent ]
maybe maybe not but
there are other countries that have had H5 pop up again and again I would think at least one of the m would have noticed. I dont think die offs of other species in H5 hotspots would go totally unoticed. Maybe im wrong

[ Parent ]
...
i think you're right...  i think it would be noticed..  i'm just not certain they wouldn't label as something else and look no further into cause. 

[ Parent ]
cats and rodents
Why arent we seeing more dead mammals in areas like Indonesia, if the virus is in the enviorment ahouldnt we see rodents feral cats [seen a few of those] and other scavenger species dying in large enough numbers to notice?

Well, we know about cats, they've been dying everywhere.  Indonesia, Iraq, Egypt.  More about them later.

But, to the extent that we are only finding about cats by looking specifically for the info in journals etc, I wouldn't expect to be able to hear about other even less obvious species like rodents to be reported in the media at all.  And unless someone goes and does a systematic survey of various small mammals, which IMHO is high time someone does that, we won't know.

Cos in underdeveloped countries, with dead chickens being thrown into garbage piles and fish ponds, unsanitary conditions are so common I wouldn't expect anybody to pay attention to whether rodents are dying!

Plus, flu viruses are very species specific.  Even in birds, they are not present in all species.  A paper recently in Emerging Infectious Diseases Susceptibility of North American Ducks and Gulls to H5N1 Highly Pathogenic Avian Influenza Viruses demonstrates that very well.

Here is a portion of the abstract:

We assessed the clinical response and extent and duration of viral shedding in 5 species of North American ducks and laughing gulls (Larus atricilla) after intranasal challenge with 2 Asian H5N1 HPAI viruses. Birds were challenged at ?10 to 16 weeks of age, consistent with temporal peaks in virus prevalence and fall migration. All species were infected, but only wood ducks (Aix sponsa) and laughing gulls exhibited illness or died.




All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
on cats specifically
This paper from the August 06 issue of Emerging Infectious Diseases (free) is very instructive.

Qinghai-like H5N1 from Domestic Cats, Northern Iraq on the various issues involved, including the difficulties of interpreting stories of cat deaths in remote locations.

Personal communications in January 2006 from field veterinarians noted deaths of domestic cats that were associated with suspected (eventually confirmed) H5N1 outbreaks in eastern Turkey (2 villages) and Kurdish northern Iraq (Sarcapcarn in Sulymaniyah Governorate and Grd Jotyar in Erbil Governorate). The clinical conditions of the birds did not suggest HPAI to villagers or consulting veterinarians. In both scenarios in Iraq, results of rapid antigen detection tests with the Anigen kit (Suwon, Republic of Korea), while positive for influenza A, were negative for H5, so the outbreaks were not thought to be caused by HPAI, but concern about the unusual deaths in cats remained. Because the regions are remote and veterinary services limited, such anecdotal reports have rarely been followed up.

The important thing to notice about this paper is that this is a Clade 2 virus, whereas previous investigations of cat infections in Thailand etc were on clade 1 viruses. 

The viruses from Iraq are most closely related to currently circulating Qinghai-like viruses, but when compared with A/bar-headed goose/Qinghai/65/2005 (H5N1) (GenBank no. DQ095622), they share only 97.4% identity at the nucleic acid level with 3 amino acid substitutions of unknown significance. On the other hand, the virus from the cat is only 93.4% identical to A/tiger/Thailand/CU-T4/2004(H5N1) (GenBank no. AY972539). These results are not surprising, given that these strains are representative of different clades (8,9). Sequencing of 1,349 bp of the N gene from cat 1 and the goose (to be submitted to GenBank) show identity at the amino acid level, and that the N genes of viruses infecting the cat and goose are >99% identical to that of A/bar-headed goose/Qinghai/65/2005(H5N1). These findings support the notion that cats may be broadly susceptible to circulating H5N1 viruses and thus may play a role in reassortment, antigenic drift, and transmission.

'Broadly susceptible' is the ominous phrase, IMO.  This means that it may be something intrinsic to H5N1, and the tendency to infect cats will remain wherever in the world the virus goes. 

Other important points on cats, what we do know:

  1. natural infections in domestic cats with suspected cat2cat transmission (14 out of 15 cats in a household infected but only 1 cat had eaten infected chicken meat) have been accurately documented in Thailand
  2. laboratory confirmation of cat2cat transmission well-documented. 
  3. tiger infection in Thailand zoo also clearly showed tiger2tiger transmission
  4. felids, ie cats, tigers, etc are previously not thought to be natural hosts for influenza viruses
  5. this would be the first mammalian group of species to show efficient mammal2mammal transmission
  6. cat infection is not confined to areas of widespread poultry outbreaks, but has been documented eg in Germany shortly after wild birds were found to be infected.  ie H5N1  does not need a land-based poultry adaptation to infect cats
  7. studies, mainly in Rotterdam, showed infected cats could excrete the virus before symptoms
  8. those infected by ingestion had virus in the nerve tissue of the gut - this is a novel route of infection for mammals
  9. virus was isolated from multiple organs, showing presence of systemic, not just respiratory disease
  10. virus attachment was mainly and in high quantities to the lower respiratory tract, and minimal in upper respiratory tract, mimicking human disease

Osterhaus and colleagues have stated clearly
http://www.nature.co...

Cats may be involved in helping the virus to adapt to efficient human-to-human transmission.

and have chided the WHO and FAO/OIE for complacency

Despite these unexpected events, the possible role of cats in the epidemiology of H5N1 virus infection has been largely overlooked by the human- and animal-health communities. As recently as 28 February 2006, a World Health Organization (WHO) press release5 stated: "There is no present evidence that domestic cats play a role in the transmission cycle of H5N1 viruses." A March press release6 from the World Organisation for Animal Health (OIE) stated: "The OIE stresses that as of today, all the natural cases in felines have not led to any change in the epidemiology of the disease that has fundamentally remained a bird disease, nor have they led to any recognized virus change in epidemiology or mutation leading to an increased virulence of the virus for felines or other mammals."

Therefore

The potential role of cats should be considered in official guidelines for controlling the spread of H5N1 virus.

Here are a few references:

  1. Feline friend or potential foe?  Kuiken et al Nature 440, 741-742 (6 April 2006)
  2. Avian H5N1 Influenza in Cats Kuiken et al, Science 8 October 2004: Vol. 306. no. 5694, p. 241
  3. FREE Influenza A Virus (H5N1) Infection in Cats Causes Systemic Disease with Potential Novel Routes of Virus Spread within and between HostsRimmelzwaan et al, American Journal of Pathology. 2006;168:176-183.
  4. FREE Qinghai-like H5N1 from Domestic Cats, Northern Iraq Yingst et al Emerging Infectious Diseases, Volume 12, Number 8-August 2006
  5. FREE Probable Tiger-to-Tiger Transmission of Avian Influenza H5N1Thanawongnuwech et al, Emerging Infectious Diseases, Vol. 11, No. 5 May 2005



    All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
and where is the WHO and FAO?
Osterhaus's comment in Nature was made in April 2006.

As of now, January 2007, the WHO still has not issued any guidelines or warnings about cats.

The FAO makes no mention of cats either in its Safety Measures or Q&A pages, nor its Recommendations on the Prevention, Control and Eradication of Highly Pathogenic Avian Influenza (HPAI) in Asia document.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
FAO H5N1 cats (and dogs and mammals) March 2006
(another undercirculated page, that should have gone to every vet and pet store and dog officer and town hall...and animal owner, and farmer...)

http://www.fao.org/A...

..."All carnivores could become infected through eating infected poultry or infected wild birds. "...

..."Recommendations

Areas where H5N1 HPAI has been diagnosed or is suspected in poultry or wild birds:

Report to the local veterinary authority any evidence of significant bird mortality both wild and domestic

Be especially vigilant for any dead or sick cats and report such findings to the local vet

Make sure contact between cats and wild birds or poultry (or their faeces) is avoided and/or keep cats inside

If cats bring a sick or dead bird inside the house, put on plastic gloves and dispense of the bird in plastic bags for collection by local veterinary animal handlers

Keep stray cats outside the house and avoid contact with them

If cats show breathing problems or nasal discharge, a veterinarian should be consulted

Do not touch or handle any sick-looking or dead cat (or other animal) and report to the authorities

Wash hands with water and soap regularly and especially after handling animals and cleaning their litter boxes or coming in contact with faeces or saliva

Dogs can only be taken outside the premises if kept on  restraint

Do not feed any water birds

Disinfect (e.g. with bleach 2-3 %) cages or other hardware with which sick animals have been transported or been in contact with.

Wash animal blankets with soap or any other commercial detergent "

...


[ Parent ]
Yes, I saw that
but I take issue with them in not putting that into their official guidance, nor the Q&A, which is where people would go to when searching for information.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
just hoping a few eyeballs find it here and pass it around
though, we know hope is not a plan...

:-/


[ Parent ]
absolutely! ;-) n/t




All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
from PFI
http://web.mac.com/m...

Monotreme has put up a series of alignments of cat and human sequences from Indonesia, showing a high degree of homology (99%) at both the nucleotide and amino acid level.  This is hardly surprising given we have been guessing this, but it still gives one a sinking feeling to see them.  Hat tip to Monotreme.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
I've been suspecting cats for some time
They can not only eat infected birds (wild or domestic) but possibly infected mice as well.  Plus, when those tigers died in Thailand after being given infected meat, didn't all the cats at the zoo end up dying?

[ Parent ]
I don't know about cats at the zoo
but not all the tigers that died were given infected chicken.

If you look at the following chart, the tigers were fed raw chickens from Oct 11.  After the Oct 16, the food was changed, but they continued to get sick all the way to Oct 28th 

Probable Tiger-to-Tiger Transmission of Avian Influenza H5N1





All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
cats and pigeons
revere has a good piece on cats and pigeons today at EM:

http://scienceblogs....

 



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
also my post at EM, on cats and pigeons
I have never been one of those who consider a pandemic by H5N1 inevitable.  But the incursion of this virus into cats as well as pigeons probably takes it one big step further in that direction. 

When the cat and tiger deaths first started in 2004, we could have said that those might be one-off events.  A couple of years later, we have reports of cat infections from multiple countries, and, more worryingly, from both clade 1, and at least 2 subclades of clade 2 viruses, assuming we haven't seen any reports of cat infections from China.  Which probably means that the ability to infect cats is not due to a rogue mutation but something almost intrinsic to this virus.

The ability to infect cats and transmission between them are both scientifically well established, although we don't know how often or how efficiently that happens.  So in terms of whether we might have an enzootic focus in urban population centers in western countries, it becomes IMHO a matter of when not if, before some infected migratory bird in Europe, for example, encounters our family cats.

The infection of pigeons, however, takes this to yet another level of risk, if it become established. One could in theory take care to avoid cats in our daily lives but pigeons and their droppings are impossible to avoid if you live in European cities.  I suspect it's not that different in the Americas. 

The bigger danger is the exposure of cats to pigeons, which will happen far more frequently.  There is indeed something almost iconic about cats and pigeons, in that, more than many other species, they both roam freely and willfully among humans in concrete jungles, but each is the nemesis to the other!  Their frequent contact is almost guaranteed.

At present, one might take some (false) comfort from the low rate of human infections compared to exposure, but this could easily change if cats instead of poultry becomes the major source of human infections.  Some speculate that is what is happening in Indonesia, but we don't know. 

For the west, if pigeon infections become widespread, then the virus is going to be among us far sooner than we are prepared for.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
Clarify the Weather Role for Me Please
It's late, I'm bushed, and I hope this makes sense...

I know that for a while we've been hearing that there will be a resurgence of H5N1 during the cold and rainy season.

And I know that H5N1 survives longer in colder temps.

My question is this...

Do these weather conditions mean

  • A surge in B2B that in turn results in a trickledown surge in human cases, or
  • A surge in human cases because the virus survives longer and is possibly more easily passed H2H?

    Thanks for any efforts to shed light.




Anyone got an answer to that one? Lisa?
I haven't had time to look.  Might do that later.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
how can these people have negative H5 test
Riahs husband has been reported to have three negative H5 tests. I find this hard to believe since his wife and children are positive. We would have to believe this man got sick at the same time his wife and children did but he got something different that just happens to have simular symtoms.

We also have another case [Randi] whom has tested positive and died. her family and neighbors also just happened to be sick at the same time she was and again have symtoms simular to H5. Yet they too test negative.

Do you happen to know the percentage of false negatives, and what factors tend to produce false negatives. We have postulated that Tamiflu can cause false negatives, is this true? Wouldnt dosing with Tamiflu also explain why some of the supposed negative patients are recovering also? Common sense tells me these negatives are not right.

We have in the past seen patients be declared negative for H5 yet later [usually after death] found to be in fact + One cluster that comes to mind was in Turkey. It only came out that they were in fact positive long after the fact in a scientific article.

Your expertise would sure clear up a lot of questions many of us have regarding these teats.
thanks


it depends on the quality and timing
of the samples taken, plus the sensitivity of the test used.

Samples that are taken too early or too late, or not stored and transported in the right conditions, will deteriorate.  A quick look at the WHO guidelineshttp://www.who.int/c... will give you an idea of what is needed.  The following chart is also from that guideline, and shows when samples are most likely to be positive.

In addition, as discussed before, PCR tests depend on having the right primers and 'probes'.  In laymen terms, these are like one half of a zipper, except that they have to match the other half exactly on every notch in order to work, so the results are very specific and therefore very accurate.

Based on known H5 sequences, scientists design these primers that will attach to specific portions of the nucleotide sequence from the H5.  But if the nucleotides at that location have changed, the test would not work.  To some extent, it's a moving target.

This article in Emerging Infectious Diseases describes the latest RT-PCR assay.  http://www.cdc.gov/n...

I haven't seen any figures for the percentage of false negatives.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
Thanks again
Again you have been able to shine some light on a confusing situation and provide information all us flu dummies can understand.

The information above certainly shows how hard it is to get accurate tests.


[ Parent ]
if you look at the timing
of when people go to hospital, and the fact that most tests are based on throat swabs, then there will likely be a lot of false negatives, unless they follow up much later with serology.  Which looks unlikely in a lot of instances.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
A question from a dummy
I've truely tried reading and understanding the indonesian thread, but about half way through a post with names, ages, hospitals, my eyes glaze over and my brain locks up.  I don't know why it's so hard for me to understand how many people and clusters we are looking at.

Could someone boil the cases in indonesia down for me?

How many clusters?

How many people in the clusters?

and

How many are in the process of being tested?

How many think this is "it"?

I'm pretty good with how much rice to buy, and how to cook it, but this stuff is just way over my head LOL

Our children change our lives, whether they live or not.
www.misschildren.org


I don't know the answer to those questions
but if it's way over your head, I would suggest just keeping an eye on the size of the biggest cluster.  The number of clusters is not half as important as that one thing.  And whether there are tertiary cases or even beyond.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
tertiary? whats that? N/T


Our children change our lives, whether they live or not.
www.misschildren.org


[ Parent ]
h2h2h
the third case in the chain.

So far, there has only been one confirmed h2h2h incident, the Karo cluster in May last year.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
Genetic susceptibility
Why Karo cluster failed to propagate beyond the family blood line?

I remember at the time, people there did not have any protective equipment, took no precautions and yet the Karo cluster failed to go further (good). The official theory was genetic predisposition of the victims.

I searched for answers on Karo clan and could not understand what made them so different, then I saw this post by SusanC and realized that 'predisposition' really meant 'susceptibility', possibly due to first cousin marriages:

http://www.newfluwik...

Since this piece of (what I think) is very important information is buried in the Indonesian news thread, I want to place it here for further discussion.  I also found some links that explain marriages and families in Indonesia:

http://www.newfluwik...

The question of genetic susceptibility has become more important since the Nature article stating that no longer needing a mutation to bind to upper respiratory tract.

You want perspective. I want perspective. Let's talk. We don't have to agree on every thing. If we do, one of us is redundant.


New research in Nature Journal
Hope someone who subscribes to "Nature" can give us more details than the newspaper articles.

From Helen Branswell:
http://www.cp.org/en...

Title- "1918 flu virus, recreated in Winnipeg, triggered overwhelming immune response"

"The virus that caused the 1918 influenza pandemic triggered an overwhelming immune response that swamped the lungs of macaque monkeys - the first primates deliberately infected with the Spanish flu virus, Canadian and American scientists reported Wednesday."....

"The research, published in the journal Nature, involved an ambitious project to painstakingly recreate the 1918 virus - only the second time this feat has been achieved."....

"The effort that led to this research began a short time later. Yoshihiro Kawaoka, a leading influenza scientist working at the University of Wisconsin, Madison, built each of the virus's eight genes from scratch, using genetic blueprints housed in a public access database."...

"It suggests if you interrupt the inflammatory chain in the innate immune response, then you might have another tool in your armamentarium," said Dr. Anthony Fauci, director of the U.S. National Institute of Allergy and Infectious Diseases"...

From another article on the same research:
  http://news.yahoo.co...

Title- "Monkey clue to how 1918 pandemic flu virus selected its targets"

"The study, which appears on Thursday in the weekly British journal Nature, is headed by Yoshihiro Kawaoka of the University of Wisconsin at Madison."...

"The answer is still hazy, but Kawaoka's team suggest a link with a gene called RIG-1, which controls a protein of the same name that directs the cascade of interferons, chemokines and cytokines.

Lab-dish tests on tissue infected with the 1918 virus showed that levels of the RIG-1 protein were lower than with tissue infected with the conventional virus: in other words, the molecular controller was somehow stopped from doing its job.

The new research amplifies work on lab mice last year that pointed the finger at an over-excited immune system, but also at activation of genes that order cells to commit suicide, a process called apoptosis."....


I'm looking at this
and the difference in pathology between 1918 and H5N1.  There's a couple of references I still need to go through, but I will post in the next couple of days.  Some interesting points worth noting.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
Statins - CIDRAP report
SusanC was promoting the news of statins (last spring)for ARDS treatment. The wheels continue to grind slowly. Now they are mentioned in regards to the new cytokine research on the monkeys. Are they going to try statins on infected monkeys? And why not infect monkeys with H5N1, and discover what treatment works best. (sorry monkeys!!)

http://www.cidrap.um...

Title -"Study sheds light on lethality of 1918 flu virus"

"One possible pharmaceutical defense strategy hinges on statins, a class of drugs used against cardiovascular disease that target the same inflammatory response observed in the flu studies. Several studies have found that patients who are taking statins experience less sepsis and bacteremia.

And a forthcoming article in the journal Critical Care Medicine follows a group of 11,400 patients with atherosclerotic disease, half of whom were taking statins, and finds that statin use cut the risk of death from infections-mostly pneumonia-by two thirds.

"This is a clinical and epidemiologic signal of protection that we ought to pay attention to, that statins are beneficial in serious infectious disease syndromes that are associated with elevated levels of proinflammatory cytokines," David Fedson, a former professor of medicine and pharmaceutical researcher who recommended exploring statins as a defense against a pandemic in a 2006 article in Clinical Infectious Diseases, said in an interview.

Unlike pandemic vaccine, he said, statins can be produced in advance-and unlike the antivirals used against flu, they are widely produced around the world in generic format, and therefore both abundant and cheap."


there is definitely a place
for more and urgent studies into statins.  Again, more later.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
294S mutation in NA in Vietnam 2005
The mutation 294S conferring tamiflu resistance was recently found in Egypt and reported in the news diary here http://www.newfluwik...

This mutation confers moderate resistance to oseltamivir (tamiflu).

It was first found in a Feb 05 from a 14-year old Vietnamese girl and published in Nature Avian flu: Isolation of drug-resistant H5N1 virus

The girl's brother (patient 2 in the chart below) was diagnosed with H5N1 infection, and she was given tamiflu 75mg once daily as prophylaxis for 3 days.  When she began to show symptoms, she was given the full dose, and subsequently recovered.

Virus isolates from this girl exhibited different degrees of sensitivity to tamiflu, and they were grown in virus culture into 10 clones (see chart).  The brother's NA was the same as clone #7, which was sensitive to tamiflu (IC50=0.6).  The H274Y mutation conferred marked resistance, as shown by the very high IC50.  The third clone has the 294S mutation, which showed moderate resistance, with slightly raised IC50.





All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


tamiflu resistant virus in Egypt - the case is open!
re-posted from the Indo thread http://www.newfluwik...

I went through another search of literature on this again.  It would seem that the only 'evidence' that N294S confers tamiflu resistance is from the Vietnamese case, described above. In that one case, the reduced sensitivity is very mild.  Since that girl recovered, and there were mixed isolates of another more potent mutation H274Y, it is hard to be certain of the actual clinical significance of N294S.

Although N294S has been cited in this review in the NEJM http://content.nejm.... I searched all the references from that review and found none on N294S, so I am assuming (which could very well be wrong) that this discussion was also based on the Vietnamese H5N1 case.

Transmission studies in ferrets http://www.journals....  showed that R292K, and E119V in N2 (H3N2) were not transmitted, but H274Y in N1 (H1N1) was transmitted.  These results tell us different mutations (and in different NA's) can result in different transmission and/or virulence, so each mutation has to be studied individually.  But I could not find any transmission or virulence study on N294S. 

In addition, I had initially assumed that the WHO announcement of finding the resistant samples was based on oseltamivir efficacy assays, such as the one above.  On reading the various press and WHO statements more carefully, I believe that was not the case.  I believe what happened was that they found the N294S mutation, did not do additional efficacy/resistance assays, and announced the finding of a resistant virus.

If what I've found is accurate, it would seem that the whole case of tamiflu resistant H5N1 in Egypt is based on the findings in the Vietnamese case, which, as I said above, is far from having established the case for clinical N294S tamiflu resistance.
 
 
 



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
Tamiflu sensitivity assays
Two methods are described in the above paper, and they are pretty standard ones. 

  1. Drug sensitivity assay. The sensitivity of the viral NA to oseltamivir carboxylate was evaluated with an NA enzyme inhibition assay based on the method of Gubareva et al.1 Methylumbelliferyl-N-acetylneuraminic acid (MUNANA, Sigma), at a final concentration of 0.1 mM, was used as a fluorescent substrate. Virus dilutions containing between 800 and 1200 fluorescence units were used in the NA inhibition assay.  Diluted virus and the drug [0.01 nM - 1 mM in 33 mM 2-[N-morpholino]ethanesulfonic acid (pH 6.0) containing 4 mM CaCl2] were mixed and incubated for 30 min at 37°C, followed by addition of the substrate. After 1 hr at 37°C, the reaction was stopped by adding 0.1M NaOH in 80% ethanol (pH 10.0). Fluorescence was quantified at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. The relationship between the concentration of inhibitor and the percentage of fluorescence inhibition was determined, and IC50 values were obtained by extrapolation of those findings.

    The normal range in this instance for sensitivity to tamiflu is 0.1 - 10nM.

  2. Animal experiments Young male ferrets (Marshall Farms, North Rose, NY) weighing 0.6 to 0.8 kg, were anesthetized with ketamine (25 mg/kg) and xylazine (2 mg/kg) by intramuscular inoculation and infected intranasally with virus (2.5 x 105 pfu).  On day 1, 3, 5, 7, or 9 postinoculation, nasal wash samples were obtained for virus titration in MDCK cells.  To evaluate the efficacy of NA inhibitors, animals were mock-treated or treated with a drug (oseltamivir, 25 mg/kg orally; zanamivir, 5 mg/kg intranasally) at 2 hours post infection and then twice a day for 5 days.




All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
Is the new Egyptian strain
being 'partially' resistant to Tamiflu, as opposed to wholly resistant, much of a consolation when the two H5N1 victims concerned died?

Would it have needed a higher dose or a swifter application of Tamiflu to have overcome that partial resistance?


see above post - we actually don't know
http://www.newfluwik...



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
prophylaxis
SusanC,
wouldn't it be better for people here to plan for prophylactic
or post-exposure-prophlaxis taking of Tamiflu or Relenza
rather than therapeutic ?
Maybe we had this, not sure. But nothing in this thread.
So basically you start taking it 5 hours after you think you might have
contacted the virus. (what dose?)And then for 2-3 days until you are sure
you have none. Do some flu-tests to determine this before onset
of symptoms. When you get symptoms increase the dose.
By that time we will know about resistance.
And don't go out while on Tamiflu and suspicious, so not to
infect others with resistant strains.
Just my conclusions, I haven't read it.

ask experts for their subjective
panflu death expectation values
and report the replies


prophylaxis is not the answer
because there just is not enough to go round. 

Secondly, the data on prophylaxis is based on seasonal flu, and there is no certainty what dose will work and for how long for H5N1. 

Thirdly, the issue of resistance still remains, ie prophylaxis becomes inadequate treatment if you are in fact infected, and you are promoting the survival of a resistant strain, which is what happened in the Vietnamese case above.  Fortunately, the resistance was not bad enough and since she was already in hospital under observations, the docs were clued up enough to immediately increase the dose, and it worked.  There is no guarantee that this would work every time.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
dengue and avian flu hard to differentiate
  This was from NAMRU-2 in the Paris anti-Avian flu conference, Institut Pasteur, 2006, on this thread at the old forum http://www.fluwikie2... Scroll down to 22:07

Herman Kosasih of NAMRU reporting on clinical and lab comparison of dengue, human flu, or avian flu in Indonesia:

H5N1 epidemiology in Indoesnia June 2005-2006

  • 532 cases: 56 confirmed + probable, 42 deaths, CFR 75%, 7 clusters
  • Median age 19, range 1.5-43 years
  • Male: Female= 1.5:1
  • 44% rural, 30% semi-rural, 26% urban
  • Most cases with poultry exposure
  • Onset to hospitalization 4.8 days
  • From hospitalization to avian influenza being suspected was 3.1 days
  • The initial diagnosis were dengue, pneumonia, typoid, and other respiratory tract infections.
  • The onset to hospitalization for dengue was 4 days, human influenza was 3.4 days, ie very similar.
  • For distinction between avian and human influenza, it was difficult until shortness of breathe happened on day 5.
  • Dengue and avian flu had similar respiratory symptoms, as well as spontaneous bleeding, so it was difficult to make a clinical distinction.
  • Complete blood count (CBC): avian flu significantly lower leukocyte, lymphocyte, and platelet than human flu, but compared to dengue, lymphocyte was lower, but platelet count was higher
  • Of the 3, H5N1 had the lowest lymphocyte count


Summary: it is difficult to distinguish AI from other endemic infections during acute illness, causing delay in diagnosis and treatment. Poultry contact, shortness of breath, serial blood counts to track leucopenia, lymphopenia, and thrombocytopenia may be useful.

Notice it took an average of 7.9 days from onset of symptoms to H5N1 infection being suspected.





All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


Recurrent misunderstandings in outbreak news reports
I have been seeing certain basic science errors being repeated by a vareity of folk when finding both bird flu and 'possibly related' stories, that I hope this comment can clear up.  Some of this will be very basic review and this is merely to bring focus to those aspects of the bugs that I want people to notice; I assume that you already know the information.

A host animal is any living thing that can be infected by another organism.  Multicellular animas can be infected by bacteria, viruses, and prions.  Bacteria can be infected by viruses, but those viruses are usually called phages. 

A host animal could be looked at like a perpetual construction site--a 'Winchester Mystery House' in the works.  It is big, it is multi-functional, it has a lot of blueprints around that are in process of being executed, and if you nip a few lots of wood off the lot, or stick an extra page into the blueprints, on a small scale there's a good chance you'll get away with it without being noticed.

Bacteria and fungi have a genome and all the mechanisms to put that genome into action.  They may have a cell nucleus, or they may not.  They have the cellular machinery to reproduce themselves given only a few, fairly simple chemistries upon which to subsist.  For example if you put them into a mixture of amino acids, most of them would be quite happy to reproduce as long as the supply holds out.

You could look at bacteria as little job shops in the vicinity of the Mystery House.  Some are harmless--they come by and pick up the construction waste from the site, cart it off, and put it to their own uses, with no harm done and perhaps even a benefit done to the House by keeping the construction site tidy.  (This is analogous to 'bacterial flora' on your skin.)  Some may be helpful--taking the waste bits and making little gifts or tools out of some of it to return to the construction site. (This is analogous to the 'beneficial bacteria' in your gut.)

But others can be bad actors.  They sneak in, take resources vital to the main project, and make off with them.  Or they come in and set up shop right in the main construction site, and refuse to leave.  (These are your pathogens.)

Now, for bacteria there are two modes for them to be found in a place.  In one mode, they are present, but harmless.  Medically, this is termed 'colonization'.  A person can be colonized by MRSA (methicillin resistant staphylococcus aureus)--they have it on their skin or in their throat--but it isn't causing them any problem.  On the "construction site", it is acting like harmless skin flora, picking off the left overs and not invading the site or taking any vital supplies.

The other mode is 'infection'.  This is where the bacteria is doing damage--it is going into the construction site and raiding it for materials or otherwise vandalizing the main project.

Now, imagine you have an infection--or, if you will, a theft on the site.  You go in to look for likely suspects.  Among the likely suspects you find that there is MRSA present--a character with a long and sordid history of bad acting.

You might be tempted to blame MRSA for the problem.  But, it may be that MRSA isn't really the culprit--that some other organism/theif is at fault, and MRSA just happened to be in the neighborhood.  Mere presence is suggestive, but does not prove, causation.

This is why you have to be careful if you have an illness and a microbe is found--in most cases the bad actor will be guilty, but sometimes it is not the infective agent, merely a colonizing agent--a bystander.

Some of you may remember the deadly hemorrhagic problem in China last year involving pig farmers and blamed on Streptococcus suis, which was found in the pig farmers afflicted.  While this remains the official explanation, no one has yet come up with an explanation as to why this formerly innocuous bacteria suddenly went killer, and there remains the possibility that some other agent caused the disease, and the S. suis was merely present on the patients because it is probably present on all pig farmers all the time, sick or not. 

This is an example of why it is important to distinguish between colonization and infection.  Colonization can make you think you have found the cause of something, when you haven't--you've 'arrested the wrong suspect'.  The problem with this is it can keep you from searching for the right suspect.

Another example of this principal is Hemophilus influenza--a bacteria isolated after the 1918 pandemic, before the discovery of viruses in 1926, and blamed at that time for the 1918 pandemic.  It causes pneumonia, and was present in many of the 1918 cases, but as we now know, it was not the cause of that scourge.

Viruses have a genome, but no active mechanism to put it into action.  Instead, they must hijack the mechanisms of life of some other organism in order to make copies of themselves.  This is equivalent to sneaking a page into the blueprints for the House.  They are completely inert and inanimate when not being used on a construction site--they are not like a little job shop  next to the construction site, but like a piece of paper litter blowing about in the breeze and getting shredded over time by the elements if not picked up b the site and put into use or storage.

Now, you can imagine that not every blueprint can be executed in every construction project.  If the 'House' is a little log cabin, and the viral blueprint calls for welded steel construction--well, that's just not going to get built on that job site, as neither the tools nor the materials are present to do it.  This is analogous to a virus which has entered a cell of a species to which it is not adapted--to an animal outside its 'host range'.  For example, due to chemical differences, it is highly unlikely for a flu virus to survive in an insect cell, unless the situation has been artificially engineered in a lab to allow that to occur.

You can also imagine that the scrap of paper blowing in the wind is going to degrade in time until it is finally unusuable as instructions in a set of blueprints.  This is analogous to a virus sitting on a surface out in the environment, where it falls apart over time.

Imagine a truck drives by, and the scrap of paper gets caught on the bumper and falls off at another construction site.  This is the role of a 'fomite'--something that carries inert virus from one place to another, but does not support replication of the virus.  When flu virus is isolated from flies, it is likely that this is the means by which the virus is present--as an inert rider on a mobile agent.

A carrier of a virus would be if the viral genome were packaged up among the legitimate cargo carried in the truck--especially if the truck were a mobile home and the virus was actively being replicated while in transit.  Chickens would be the equivalent, for H5N1 flu.

I bring this up because there have been findings of H5N1 flu virus in all kinds of unlikely critters and locations, and people have been getting upset by the thought that the virus infects everything.  It doesn't *infect* everything, sometimes it is just an inert passenger.

Prions, for those interested, are proteins that are mis-folded and cause mis-folding of other proteins.  They have no genetic material.  Prions, by this analogy, would be like a right-handed mold for making molds, that gets into a left-handed mold shop.  Over time, right handed molds get into the system, and before long, that 'left handed' shop is making both right and left handed molds and the whole system comes tumbling down.

medical information provided is for discussion purposes only and should not be construed as medical advice. if you believe you have a medical problem, consult your practitioner.


Splain sequences that are avian verses human please
Susan you had to know this one was coming :-)

Can an human sequence infect birds? Or vice versa?

What is the difference between a avian sequence and a human sequence anyway?

Lastly what is the significance of a sequence being human or avain? ie what does it mean where the rubber meets the road?

Thanks again!
janet


these are good questions
It's important to get the basic concepts down.  There is sometimes a slight confusion in terminology, cos when we say an 'avian sequence' we could mean that it was taken from an avian source, or we could mean that it was something very similar to what one would expect from an avian source, but it could have been taken from a human sample.

Influenza A viruses mutate and adapt to whichever host it gets into.  However, that adaptation is not a one-step immediate thing, it's something that happens over time, perhaps over many host2host infections.  When the virus first infects a new species, eg H5N1 b2h, the samples that you found from human subjects would have evolved somewhat from the avian samples, but they have not gotten far enough yet and can still be distinguished from those that are fully adapted to humans.

We have in GenBank and other places data on many sequences done for many different samples over many years.  These are all labelled with where they were taken, from which species etc.  If you line up all the ones known to be from birds, you find that they are all very similar but there are also many differences between them.  A phylogenetic tree is a way to 'map' those differences, so that those that are most similar are placed nearest each other, and those that are different would be further apart. 

You can do the same for sequences known to be taken from humans, and compare them to each other, and to the avian sequences in a similar way. 

In the case of seasonal flu, ie the virus having already become well adapted to humans over many years, on a phylogenetic tree, they would be grouped close together but all of them would be distinctly separate from avian sequences.

In the case of an avian virus that has recently jumped species b2h, a sample taken from a human would still appear on the phylogenetic tree within the avian branches. 

Where a sequence is placed can give vital clues as to the origin of the virus.  To give one example, there have recently been 2 deaths from (apparently) seasonal flu H3N2 in Thailand in young subjects.  The Thai government has asked for outside help to sequence the autopsy lung samples to see if there are any significant changes.  What they did not say of course was what they were looking for.  Suppose they discover that out of the 8 gene segments, there are 1 or 2 that do not fall within the human subclades (or branches) but within let's say avian ones.  That's a big red flag for a reassortment event having happened and created a new strain!



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
Thanks again! n/t


[ Parent ]
Genome match for two patients in Indo
Monotreme is reporting two patients who were unrelated and lived in different areas have genome matches on all 8 genes. He is saying this means they contracted the virus from the same source. Is this true? Enlighten us please as to what this means or doesnt mean

Thanks as always
janetn


exact matches!
Well, I would defer to Monotreme on that, cos he knows a lot more about the esoterics of genome matches than I do.  I certainly trust his judgment on these matters!

I don't know how conserved these viruses can be.  We've had this discussion before, and I never got clear on what exactly are the chances of all 8 gene segments being exactly the same.  Certainly if they live in different geographical areas it would require some investigation.

There is also the possibility of laboratory error, although again not being a lab scientist, I wouldn't be able to tell you how likely that is!



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
Same hospital - possible sample mix up?
http://www.newfluwik...

both had no direct contact with poultry

http://www.newfluwik...

Father suspected slaughter house around Ani's house, but too far?

http://www.newfluwik...

You want perspective. I want perspective. Let's talk. We don't have to agree on every thing. If we do, one of us is redundant.


[ Parent ]
hospital mix up
is certainly possible.

Sadly, it is commoner than you would think, even in the best hospitals...



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...


[ Parent ]
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