Anti-viral resistance in 2009 A/H1N1 flu - historical compilation of news

[St Michael]

Re: Taiwan: woman died of H1N1; Tamiflu resistance investigated

Two more deaths, one shows Tamiflu resistance

Tuesday, August 25, 2009
The China Post news staff

Health authorities yesterday confirmed two more A(H1N1) related deaths, including one victim who may have shown resistance to the antiviral drug Tamiflu, local media reported.

The patient died from the new virus less than one week after showing signs of the disease, authorities said.

Officials from the Centers for Disease Control (CDC) indicated that at present the agency cannot confirm the reason behind the ineffectiveness of the medication, which could have be the result of drug resistance or mutation of the A(H1N1) virus.

The case involved a 44-year-old woman from Pingtung County who experienced discomfort on Aug. 17 and later came down with a fever of 39 degree-Celsius on Aug. 20, the CDC said.

The fever was also coupled with severe cough, shortness of breath, which developed into a respiratory infection on Aug. 21, the CDC added.

The woman was not a Typhoon Morakot victim, said the CDC.

Officials were cited as saying that the woman had tested positive for the flu upon taking the rapid influenza diagnostic test and was placed on a course of Tamiflu treatment.

The medication did not help the woman, who died from multiple organ failure, the CDC said.

The other death involved a 6-year-old boy in Changhua County who came down with cough, runny nose, sore throat and high fever on July 19, said the agency.

Officials noted that he initially sought medical help from a local clinic and did not take the rapid test nor was given antiviral drugs.

He was later transferred to a larger medical facility before his illness took a turn for the worse on July 27, after which he was under intensive medical care until passing away on Aug. 21, said officials.

The two deaths put the current number of fatalities at five, involving cases from Taipei City, Kaohsiung City, Taitung County, Changhua County and Pingtung County, the CDC said.

Five patients with serious A(H1N1) infections remain in ICUs, said officials.

Typhoon Relief Soldiers Confirmed with A(H1N1)

The Department of Health (DOH) last night reported two cases of massive flu outbreaks involving troops participating in Typhoon Morakot rescue efforts in southern Taiwan, with four of the soldiers confirmed as carrying the A(H1N1) virus and another as carrying the H3N2 seasonal strain.

Steve Kuo, head of the CDC, dismissed rumors that two soldiers had succumbed the A(H1N1) flu.

Given the grave epidemic situation, Premier Liu Chao Shiuan instructed all soldiers to wear face masks in devastation areas as an extra level of protection.

Volunteers in disaster zones are also required to wear them, added the paper.

Health authorities previously reported that the new flu strains tend to attack the young and healthy, who do not seem to be equipped with the antibodies to fight the disease.

Liu's orders marked the first time the government has mandated the wearing of face masks since the country recorded its first A(H1N1) case, said the United Evening News.

Meanwhile, 300 people in Wan-nei village, Pingtung County have fallen ill, with many coming down with fever, said local media.

Health officials have momentarily ruled out the novel flu virus as the culprit behind the illnesses in the village of Wan-dan Township and believed leptospirosis to blame as animal carcasses have been left scattered about pending disposal after floodwaters receded.

Leptospirosis, or Weil's disease, is a bacterial infection commonly transmitted to humans via urine-contaminated water coming into contact with unhealed breaks in the skin or with mucous membranes.

Taiwan is still recovering from the destruction wrought by Typhoon Morakot, and Liu said epidemic control measures should be given priority at emergency shelters for people left homeless by the natural disaster, according to CNA reports.

As of Monday, more than 6,000 survivors remain in shelters in the hardest-hit zones in southern Taiwan's Pingtung, Kaohsiung and Chiayi counties, said the CNA.

Premier Ordered Bulking Up Antiviral Drug Supply

Premier Liu Chao-shiuan said the government will expand procurement and boost current stock supplies of antiviral drugs Tamiflu and Relenza, from a reserve that can supply the needs of 18 percent of the population to one that can meet demands of 30 percent.

Yaung Chih-liang, the head of the Cabinet-level health department, noted the CDC will also relax the criteria by which patients are given antiviral medication, enabling doctors to exercise their own discretion as opposed to solely relying on results of rapid tests.

Those with underlying illnesses and recent contact with A(H1N1) patients, pregnant women and children under five will be given priority, Yaung added.

According to CNA reports, The Central Epidemics Command will soon convene an emergency meeting to discuss increasing the country's capabilities to fight the epidemic.

In response to the spike in the number of infections, the Ministry of Education (MOE) yesterday pointed out the need for schools to closely monitor students' temperatures as well as homeroom teachers to keep careful watch for flu-like symptoms such as fever amongst children.

Faculty should take an active role in extending care to students as well as in educating hand or respiratory hygiene, said the MOE.

Given continued outbreaks of the disease, the government's strategy will be put to the test after classes resume next week, said health experts.

In related news, another school in Taipei City suspended classes, marking the 15th academic institution to do so since Aug.1, city government officials explained.

Daan Junior High School said two students in the ninth grade were confirmed as carrying the A(H1N1) virus and another two exhibited flu-like symptoms.

According to CNA reports, the number of collective A(H1N1) infections is believed to have crossed the 100 mark, and more than 10,000 infections are recorded every week based on CDC calculations.

[HenryN]

Re: Sequences from Tamiflu Resistant Immunocompromised Patients Released

You asked this before. I don't have any inside info so I'll venture a guess that the answer is "no".

Since they were testing backwards and the July 28 sequence was the one announced to be resistant and the one to be released, a person might assume the others tested sensitive.

Unless, of course, there is some reason for the CDC to keep the earlier results secret (if those also showed resistance)?

July 14 had H274Y.

[HenryN]

Re: Sequences from Tamiflu Resistant Immunocompromised Patients Released

Here's a summary I did on the symptoms, testing, treatment, etc for the 2 cases. There is a big difference between them; maybe this is reflected by the differences in the sequences.

1. 18 year old male A/Washington/28/2009/07/14,m18
On April 29, 2009, he was hospitalized for a hematopoietic stem cell transplant, which he received on May 7. He received immunosuppressive treatment prior to his transplantation and remained hospitalized in a single-patient room after the transplantation.
May 31: he developed symptoms and tested positive
June 1: started a 10 day treatment of Tami
June 4: virus was again detected in a viral culture
June 4: Tami was extended for 20 days ~ June 20
June 7: patient was discharged
June 11: virus again detected in a viral culture
July 7: nasal wash specimen tested positive
July 8: he was started Tami again
July 14: became ill with a fever & staph infection
July 14 tested positive for panflu, Tami was increased to a high dose, 150 mg orally, twice a day This is the collection date of the released sequence
July 16: he had increased rhinorrhea and mild cough
July 18: he was discharged still taking oseltamivir
Aug 6: CDC testing on a June 4 specimen showed susceptibility but July 30 specimen showed resistance.
Aug 6: Tami was stopped, no further treatment, patient was assmptomatic
Aug 10/11: testing on a May 31 specimen showed susceptibility. June 11 & July 14 specimens showed resistance.

2. 47 yo female A/Washington/29/2009/07/28,f47
She had a previous hematopoietic stem cell transplant for leukemia and had a recurrence of leukemia in December 2008. She underwent two cycles of immunosuppressive chemotherapy during March--April 2009. On June 21, she was admitted to the hospital for further chemotherapy; she also had developed a fever and symptoms of an upper respiratory infection. The patient's hospital course was complicated by prolonged neutropenia and protracted bone marrow recovery, neutropenic fever, coagulase-negative Staphylococcus bacteremia, and Pneumocystis jirovecii pneumonia.
June 21: she developed symptoms.
June 21: test result was indeterminate because of an inadequate cellular specimen
June 26: tested positive for influenza A
June 26-July 1: Antiviral treatment with high-dose oseltamivir (150 mg orally, twice a day) and rimantadine (100 mg orally, twice a day)
July 3: the viral isolate was identified as novel influenza A (H1N1), and high-dose oseltamivir and rimantadine were restarted.
July 3: The patient's respiratory status worsened, and she required supplemental oxygen for hypoxia
August 4: treatment with inhaled zanamivir was attempted, but was poorly tolerated, and oseltamivir was continued.
Aug 4: testing showed positives for novel flu on samples from July 6, 14,16 & July 28 This is the collection date of the released sequence
August 6: the June 21 sequence did not have the H275Y mutation but was detected in the July 28 specimen.
Aug 6: Treatment of the patient with oseltamivir was discontinued when results became available and treatment with inhaled zanamivir after identification of oseltamivir resistance again was attempted but poorly tolerated.
Aug 7: intravenous zanamivir and aerosolized ribavirin therapy were initiated.
August 10: CDC received other previously collected virus isolates from this patient for testing, and pyrosequencing of a virus isolated from a specimen collected on July 14 had the H275Y mutation,
August 13: the patient remained symptomatic and hospitalized on intravenous zanamivir and had been switched to oral ribavirin because of intolerance of aerosolized ribavirin.

So was H274Y detectable in the July 3 and July 6 collections from 47F?

[Flufreak]

Re: Sequences from Tamiflu Resistant Immunocompromised Patients Released

Commentary<o:p></o:p>

"Matching Sequences fromTamiflu Resistance Patients in Seattle
Recombinomics Commentary 11:11
August 24, 2009

Of the four oseltamivir resistant 2009 influenza A (H1N1) viruses, two were original clinical samples detected by the neuraminidase inhibition assay, and two were viral isolates detected by sequence analysis of a neuraminidase gene.

The above comments from the latest CDC update describe the four reported examples of pandemic H1N1 oseltamivir resistance in the United States. The four isolates are from two clusters. One cluster involves two immuno-suppressed patients in Seattle who were not epidemiologically linked, as well as two summer campers in North Carolina who were taking prophylactic Tamiflu because of a pandemic H1N1 outbreak in the camp. These two sets of clusters suggest the resistance is not due to independent spontaneous events.

The CDC has released the HA and NA sequences from the two immune-suppressed patients. Both NA sequences have a defining polymorphism (A1230G), in addition to H274Y (encoded by C823T), which also supports the presence of a minor species circulating with this sub-clade, and raises concerns that the resistance associated with this sub-clone is widespread.

The public sequences are from the 18M patient with a recurrence (A/Washington/28/2009) and 47F (A/Washington/29/2009), who remains hospitalized after treatment with Tamiflu, Relenza, and Ribaviron. Although initial samples collected from both patients were Tamiflu sensitive, the isolates collected significantly after the start of Tamiflu treatment had H274Y. Although both had clean signals for C823T (which encodes for H274Y), both sequences had mixed signals at other positions. Both sequences also had addition changes that were not in common, indicating those changes were acquired after the acquisition of A1230G. However, the linkage of A1230G and C823T may have been established in a minor population at an earlier date, raising concerns that H274Y is widespread. A1230G is present in multiple isolates from Washington state (see list here), but is also present in multiple isolates from Mexico and Nova Scotia. as well as sequences from Oregon, Florida, China (Jiangsu and Guangdong), New Zealand, and Singapore.

The concept silent circulation of minor populations with H274Y is also supported by the two cases from the summer camp in North Carolina. Although sequences from the two campers haven't been released, the clustering of two cases in campers at the same location at the same time further supports a minor species. Two independent events are unlikely since both patients were attendees of the same camp. Moreover, the two cases in July were followed by rapidly spreading outbreak in schools in August in adjacent regions (see map), raising concerns that H274Y is also widespread in the area and contributing to the rapid spread among students in the area.

Sequence data from the campers, as well as students in the area would be useful."

This may be off topic, and already answered, but does H274Y increase transmission among those NOT treated/ prevented with Tamiflu?

[Treyfish]

Re: DOH to increase Tamiflu use

国内每周新增近万名新流感病例，卫生署因应疫情扩大，研拟放宽?克流感?使用时机，病患仍需先接受快速筛检 ，不过快筛试剂现在却缺货。

Domestic per week were nearly ten thousand new cases of influenza, the Department of Health in response to spread of the disease, to develop relaxation "Tamiflu" using the time, the patient still need to first accept the rapid screening, but screening reagents now out of stock fast.

(图为克流感，中央社资料照片)

(The photo, Tamiflu, CNA Data Center)

台克流感拟扩大给付快筛试剂却缺货

Taiwan intended to extend payment of Tamiflu fast screening reagents are out of stock

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【大纪元8月23日报导】(中央社记者陈清芳台北23日电)台湾每周新增近万名新流感病例，台湾卫生署因应 疫情扩大，研拟放宽?克流感?使用时机，病患仍需先接受快速筛检，不过快筛试剂现在却缺货。

By August 23 report (PG's 23 electric Chen Qingfang Taipei) Taiwan ten thousand people per week added a new influenza cases, Taiwan's Health Department in response to spread of the disease, development of relaxation "Tamiflu," using the time, the patient still first undergo rapid screening, but the fast screening reagents now out of stock.

?克流感?于15日列入健保给付，快筛试剂则由卫生署疾病管制局负担、健保局垫付。

"Tamiflu" was included in the National Health Insurance payments on the 15th, fast screening reagents from the burden of the Department of Health Bureau of Disease Control, National Health Insurance Bureau advance.

卫生署长杨志良昨天指出，将于近期开会讨论扩大给付?克流感?。

Department of Health Director Chih-Liang Yaung pointed out yesterday, will soon meet to discuss expansion of pay "Tamiflu."

湾疾管局长郭旭崧今天透露，快筛试剂对流感阳性反应的敏感度为3到7成，其差异因病情阶段、病毒量及采检方 式而异，目前专家认为即使快筛阴性，但经临床医师诊断流感且病程迅速或有肺炎者，可自费用?克流感?，预计 不久之后，这些病人可获健保给付使用?克流感?。

Chang Hsu-Sung Kuo Taiwan CDC said today, fast screening reagents for influenza positive had a sensitivity of 3-7 into their different stages of his illness, the virus load and mining-location to another, now experts believe that even rapid screening negative, but after clinicians and the duration of the rapid diagnosis of influenza or pneumonia, may be from the cost "Tamiflu", is expected soon, these patients will receive payments for the use of "Tamiflu."

郭旭崧认为，如果全面放弃快筛、完全不作检验即对类流感病患投以?克流感?，一来可能会造成滥用，助长流感 病毒的抗药性，二来，未染流感却使用?克流感?，有3成会出现副作用。

Hsu-Sung Kuo believes that if fully abandon the quick screen, totally not inspected the right to vote influenza-like illness as "Tamiflu", a move could lead to abuse, contribute to resistance to influenza viruses, and secondly, it is not stained influenza use "Tamiflu "There are 3 into the side effects will appear.

不过，?克流感?及快筛试剂二者兼备的医疗院所集中在大型医院，部分诊所或小医院?克流感?进货有限，更未 采购快筛试剂，以致于上门的类流感病患被迫转诊，必须到大医院，快筛确定为A流感阳性者，才可使用?克流感 ?。

However, "Tamiflu," and the fast screening of medical reagents between the two institutions both concentrated in large hospitals, some clinics or small hospitals "Tamiflu" purchase is limited, even non-procurement of rapid screening reagents, resulting in flu-like illness at the door has been forced referral, have to go to big hospitals, fast screen identified as influenza A positive, can use "Tamiflu."

疾管局人员指出，目前快筛试剂的单价约250到300元，数量不足，主要原因是业者生产量少，单价成本逼近 健保给付的300元，业者的利润不高。

CDC officials noted that the current rapid screening reagent is about 250-300 yuan price, quantity shortage was mainly due to less production of the industry, price the cost of health insurance payments close to 300 yuan, the industry's profit is not high.

疾管局长郭旭崧和中央健保局总经理李丞华说，卫生署正协调长庚生技等业者，赶快增产快速筛检试剂，以免开学 之后，类流感病患涌现，届时将供不应求。

CDC Long Hsu-Sung Kuo, and Cheng-Hua Lee, general manager of the Central Health Insurance Bureau, said the Department of Health is coordinating Venus and other biotech industry and rapidly increasing production rapidly screening reagents, so as to avoid school after the emergence of influenza-like illness, will be in short supply.

美东时间: 2009-08-23 06:47:44 AM 【万年历】

EDT: 2009-08-23 06:47:44 AMhttp://74.125.113.132/translate_c?hl...c4Svnx58kD8HnA

[Gert van der Hoek]

Tamiflu resistance in pandemic influenza - historical compilation of news

08/24/2009

Two more deaths from H1N1

The Center for Disease Control (CDC) announced two new fatalities from the H1N1 flu. This raises the death count from the disease to 5.

The two new deaths were one 6-year-old boy and a 44-year-old woman. The boy died after more than a month in the hospital. But the woman died a week after falling ill.

The rapid progression of her illness despite the administration of anti-viral medication raises fears that she might have a strain of H1N1 that has developed some resistance to the medication. CDC Director Steve Kuo explains.

"This is why we have to have a special investigation of this death," said Kuo, "we need to retrace the steps that lead up to the fatality to figure out whether the drug was given too slowly or whether the virus was able to resist the medication."

Kuo said severe H1N1 cases has increased to 43 with 5 fatalities. Five of them are in intensive care. Around the world, almost 1,800 people have died from swine flu as of the 24th. America suffered the most deaths, with 477 deaths, followed Argentina with 404 deaths. In Asia, Thailand has suffered the most serious losses, with 111 deaths.

The Ministry of Education (MOE) has advised schools to measure the temperatures of returning students and for teachers to watch out for flu symptoms. The spread of H1N1 will also affect Morakot disaster relief efforts. All volunteers and military relief workers now have to wear facemasks. CDC official says H1N1 disproportionately affects younger people, and most of the volunteers are young.

Thirteen members of the military involved in disaster relief efforts have fallen ill from H1N1 according to early testing results. There has been reports of residents in Pingtung’s Wandan (??) county coming down with flu-like symptoms, including running fevers. Kuo said the CDC’s investigations have ruled out H1N1 but the true cause of the illnesses remains unclear.

In response to the continuing H1N1 situation, the premier, Liu Chao-shiuan has taken the advice of experts and made the retroviral medication Tamiflu available to all patients suffering from influenza complications, whether or not tests show they have H1N1. The cost of the drugs will come out of the general budget rather than National Health Insurance funds.

http://english.rti.org.tw/Content/GetSingleNews.aspx?ContentID=85237

[mixin]

Re: Sequences from Tamiflu Resistant Immunocompromised Patients Released

Here's a summary I did on the symptoms, testing, treatment, etc for the 2 cases. There is a big difference between them; maybe this is reflected by the differences in the sequences.

1. 18 year old male A/Washington/28/2009/07/14,m18
On April 29, 2009, he was hospitalized for a hematopoietic stem cell transplant, which he received on May 7. He received immunosuppressive treatment prior to his transplantation and remained hospitalized in a single-patient room after the transplantation.
May 31: he developed symptoms and tested positive
June 1: started a 10 day treatment of Tami
June 4: virus was again detected in a viral culture
June 4: Tami was extended for 20 days ~ June 20
June 7: patient was discharged
June 11: virus again detected in a viral culture
July 7: nasal wash specimen tested positive
July 8: he was started Tami again
July 14: became ill with a fever & staph infection
July 14 tested positive for panflu, Tami was increased to a high dose, 150 mg orally, twice a day This is the collection date of the released sequence
July 16: he had increased rhinorrhea and mild cough
July 18: he was discharged still taking oseltamivir
Aug 6: CDC testing on a June 4 specimen showed susceptibility but July 30 specimen showed resistance.
Aug 6: Tami was stopped, no further treatment, patient was assmptomatic
Aug 10/11: testing on a May 31 specimen showed susceptibility. June 11 & July 14 specimens showed resistance.

2. 47 yo female A/Washington/29/2009/07/28,f47
She had a previous hematopoietic stem cell transplant for leukemia and had a recurrence of leukemia in December 2008. She underwent two cycles of immunosuppressive chemotherapy during March--April 2009. On June 21, she was admitted to the hospital for further chemotherapy; she also had developed a fever and symptoms of an upper respiratory infection. The patient's hospital course was complicated by prolonged neutropenia and protracted bone marrow recovery, neutropenic fever, coagulase-negative Staphylococcus bacteremia, and Pneumocystis jirovecii pneumonia.
June 21: she developed symptoms.
June 21: test result was indeterminate because of an inadequate cellular specimen
June 26: tested positive for influenza A
June 26-July 1: Antiviral treatment with high-dose oseltamivir (150 mg orally, twice a day) and rimantadine (100 mg orally, twice a day)
July 3: the viral isolate was identified as novel influenza A (H1N1), and high-dose oseltamivir and rimantadine were restarted.
July 3: The patient's respiratory status worsened, and she required supplemental oxygen for hypoxia
August 4: treatment with inhaled zanamivir was attempted, but was poorly tolerated, and oseltamivir was continued.
Aug 4: testing showed positives for novel flu on samples from July 6, 14,16 & July 28 This is the collection date of the released sequence
August 6: the June 21 sequence did not have the H275Y mutation but was detected in the July 28 specimen.
Aug 6: Treatment of the patient with oseltamivir was discontinued when results became available and treatment with inhaled zanamivir after identification of oseltamivir resistance again was attempted but poorly tolerated.
Aug 7: intravenous zanamivir and aerosolized ribavirin therapy were initiated.
August 10: CDC received other previously collected virus isolates from this patient for testing, and pyrosequencing of a virus isolated from a specimen collected on July 14 had the H275Y mutation,
August 13: the patient remained symptomatic and hospitalized on intravenous zanamivir and had been switched to oral ribavirin because of intolerance of aerosolized ribavirin.

[AlaskaDenise]

Re: Sequences from Tamiflu Resistant Immunocompromised Patients Released

Discussions about tamiflu resistance and immunocompromised patients can get very difficult for the average FT reader to understand. We'd all appreciate it if those posters with scientific backgrounds would do more "translation" into everyday terms.

What's being presented to the public about this topic is often mistranslated by media, so your understandable explanations are badly needed.

After 4 years of trying, I understand a little, but still get quite lost when the jargon gets too deep. I get totally lost with gsgs's statistical data.

While we can appreciate your desire to have scientific discussion, please remember what role that FT has played in your scientific pursuits, specifically, in post #54: "The story began when I was collaborating with NAMRU-3 on H5N1 outbreaks in Egypt." We'd be grateful if you could express your appreciation by phrasing your scientific discussions in terms we could all understand and other appreciation might also be in order.

.

[HenryN]

Re: Sequences from Tamiflu Resistant Immunocompromised Patients Released

Two questions:

In addition to the Gharbiya chicken, can you point to 2 or 3 additional examples of SNP dual infection changes within a single individual also supported by subsequent testing?

What is your direct response to pjie's 2b hypothesis? S/He thinks your proposition is very unlikely, possibly based on his/her prior observations and training.

Would you present here a <FOCUSED, limited>response to the question being posed and your immediately prior answer?

To get the data like the Gharbiyah clusters requires cloning and dsiclosure of full sequences, which generally isn't done in influenza. However, cloning data (bits and pieces represnted by indovidual polymorphisms has been shown in Tamiflu resistance (sister of patient who who holds the record for recover after something like 90 days of hospitalization. She was in prophylactic Tamiflu and had H274Y and N294S (as well as other changes). Similar mixing and matching in combinations of recenptor bind domaun changes in H5N1 in Vietnam. Both papers by Kawaoka and both papers withheld full sequneces of clones (where recombination would be OBVIOUS).

On the second question, the skepticism is based on the misconception by non-virologists that dual (or co-infections) are rare. This misconception is supported by "clean" sequences. However, the origin sample/sequence is quite "dirty" and most sequences are cleaned up by cloning prior to publication (and more "dirt" is swept under the rug by "consensus" sequences. As seen in Gharbiya, for 10 of the 11 positions, 1/4 of the positions were the "other" polymorphism, which "disappeared" in the consensus sequences (which is all that is made public).

[HenryN]

Re: Sequences from Tamiflu Resistant Immunocompromised Patients Released

Hmm, there's a subtlety here I hadn't appreciated previously, and I'd like it if you could clarify matters. When you say that recombination is widespread, do you mean that a single patient gets infected by two different flu strains which then recombine, or (bearing in mind that flu transmits as a population rather than a single sequence) do you mean that there is recombination between the various sequences comprising a single infection?

Toy model here, let's have a single strain A which splits into substrains B and C. Some time after B and C have split, a given polymorphism X appears in both of them. Three possibilities:

1) The mutation X occurs twice independently in substrains B and C

2a) Mutation X occurred once on branch B. Subsequently a patient became simultaneously infected with substrains B and C, and the polymorphism recombined onto C.

2b) Mutation X occurred once in A, the common ancestor of B and C. It was not detected because it was at low levels and didn't show up in the consensus sequence. After B and C have diverged, each of them still retains a low proportion of sequences carrying polymorphism X. Subsequently X is able to separately spread to fixation within B and C, because polymorphisms are freely mobile within each individual population.


If I understand you rightly, you're saying that 2b is the major scenario. That would have significant and testable implications. In particular:

* Since the mutation is deduced to have occurred in A, all other branches from A are at risk of acquiring polymorphism X, whereas strains branching off earlier are at much less risk of acquiring X. Thus you can predict which subclades are more likely to acquire a given change.

* If you could sequence all the individual virions from a given individual, scenario 2b looks like it should make fairly strong predictions about the distribution of polymorphisms between the individual virions. For example, if you had three polymorphisms with a minor allele frequency of 1/10, then 1/1000 of the virions should have all three polymorphisms. This is not what you'd predict from a random mutation hypothesis.



In genetic terms, what scenario 2b would mean is that within a single infected individual, flu should be modelled as a panmictic, freely recombining population. Transmission between patients is equivalent to a severe population bottleneck, with a subset of the flu population being passed on. Once transmission has occurred, there is then a geographic barrier to further "interbreeding" between the daughter populations, in that they're now existing in separate people! If someone gets infected by two strains, it's the equivalent of breaking one of those geographic barriers and re-merging two populations, which may or may not subsequently be compatible enough to recombine with each other. Dual infection seems likely to be rare (but more likely in epidemic / pandemic conditions).


Can you confirm that I've understood your model correctly? Panmixis between the individual sequences present in a single infection is a lot easier to grasp than the nebulous "everything recombines with everything" vibe I get from many of the posts here (not necessarily yours).

The dual infections are quite common and I can give you a specific example with real plaque purification (cloning) to show that recombination really does happen and multiple species can be generated involving the exchange of SNPs.

It was a rather dramatic example of many of the points I have been making, and some of the results have been described

http://precedings.nature.com/documents/459/version/4

However, the data that would make you a "believer" in recombination in influenza has been block by politics (of the worst kind), but the data are quite real.

The story began when I was collaborating with NAMRU-3 on H5N1 outbreaks in Egypt. They had been only sequencing HA and I suggested that they expand the sequencing effort and the highest prioity would be NA. The timing was good becasue it was the end of 2006 and one of the first NA sequences generated was for the Ghrabiya cluster, and the NA sequence showed tamiflu resistance. It wasn't the common H274Y that has been discussed here, but was N294S, which produced somewhat weaker resistance, 10-20 fold instead of 300-1000 fold seen in H274Y. However, 10-20 fold was more than high enough for treatment problems becasue most treatments are borderline, so a requirement for 10-20X more Tamiflu would render that approved treatment level useless.

However, the remarkable aspect of the resistance was the fact that N294S was present BEFORE treatment raising concerns that the polymorphism was on a fit H5N1 and was widespread in birds. As a result, NAMRU-3, which usually focused on human cases, was getting more poultry samples. A fairly extensive seach failed to find N294S in birds or patients, but on Feb 15, a Gharbiya-like sequence was identified in a chicken in Gharbiya, but it didn't have N294S

http://www.ncbi.nlm.nih.gov/nuccore/EF486246?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum

However, the NA sequence was a mixture and one position was represented with a Y and that position was polymorphic in the H5N1 sequences in Egypt (other isolates had both "C" and "T" at that position), including two other chicken isolates from Gharbiya that had 11 differences in the NA sequence.

The same was true for the HA sequence

http://www.ncbi.nlm.nih.gov/nuccore/EF469660?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum

which had three positions that were represented with a "R" and all three positions were polymorphic with multiple isolates haveing A or G at all three positions, including the same two isolates from Gharbiya collected at the same time (and these two sequences, which were identical to each other had 22 differences the Gharbiya HA sequences).

However, although the three Gharbiya chicken sequences fell into two distinct groups (11 NA differences and 22 HA difference) both had picked up a synonymous NA polymorphism, G743A, which had never been seen in any prior H5N1 in Egypt. The acquistion of the same polymorphism at the same place and same time on two very distinct backgrounds (both backgrounds have been indentified earlier, based on NA and HA sequneces), was a strong signal for recombination.

In addition, the multiple mixed signals in the HA and NA sequences raised the possibility that the Tamifu resistance (N294S) was lurking below the detection level, and could be found by cloning and sequencing the clones.

Consquently the sample from the chicken with the mixture that was related to the Gharbiya cluster was plaque purified and 44 NA sequences were generated. The clones showed that the chicken was indeed infected with two major species that were in a 3:1 ratio. The dominant sequence was closely related to the Gharbiya cluster sequence, but none had N294S, and all had G743A. The minor species matched the other two chicken sequences and also didn't have N294S (as expected), but all of the minor species clones also had G743A, demonstrating that the same change had indeed been acquired by two different backgrounds, and not only were in the same place at the same time, but were also in the same chicken.

Moreover, a few of the sequences were recombinants, with 9 or 10 of the 11 positions matching one parental sequence and 1 or 2 matching the other. Thus, the plaque purified clones demonstrated recombination involving the exchange of one or two SNPs.

However, the data also said quite a bit about what was seen in a mixed sample. Even though the two NA sequences had differences at 11 positions, only 1 of the 11 showed up as a mixed signal in the "consensus" sequence. One of the plaque purified clones was still a mixture, which appeared to be a 50/50 mixture, becasue sequencing of that sample gave mixed signals at all 11 positions. However, in the uncloned sample, 10 of 11 positions were "clean" even though 1/4 of the sequences in the sample were the "other" sequence which was not present in the "consensus".

Thus, there was clear recombination in the Gharbya chicken with the Gharbiya cluster sequence, and the vast majority of the mixed positions (10/11) did not give a mixed signal in the "consensus" sequence.

As seen in the first link above, the G743A synonymous polymorphism subsequently appeared on multiple H5N1 backgrounds in Egypt, as well as very different clade 2.2 H5N1 backgrounds in Russia, Kuwait, Ghana, Ivory Coast, all at the same time (ealry 2007).

Recombination involving the acquisition of SNPs is quite real in infleunza and quite common.

[HenryN]

Re: Sequences from Tamiflu Resistant Immunocompromised Patients Released

Thanks for the clarification. I was actually writing the post you just answered while you were offering the clarification to Mamabird, so it was already mooted by the time I hit submit.

Here is a bit more detail on how the polymorphisms are ordered and why the finding of H274Y on two isolates on the same branch signal a population that already had H274Y PRIOR to treatment (but the sequences in the database are "consensus" sequences of cloned isolates, and both approaches would "erase" or hide the presence of H274Y. If 10&#37; of the viruses had H274Y, it would not appear in the "consensus" sequence (the level ahs to be in the 30-50% range to appear as a mixed signal - which would be a Y for H274Y where a "C" is changed to "T" (Y would mean that both or present). Similarly 90% of the clones would be wild type, so unless multiple clones were sequenced, most of the time the wild type would be generated (and of course if the H274Y was in only 1%, it would be even harder to detect, unless it was selected by Tamiflu treatemnt, and the sample was collected after the start of treatment, like Washington (or the two campers in NC). The fact the 4 confirmed cases of resistance in the US were in 2 clusters of 2 alone points to a pre-existing sequence, because if it is "spontaneous" there is no reason to cluster in time and space.

The travel log presnted is for one change (A1231G) that is in 22 sequences (from 21 isolates). Thus, this is a change thet happened early (April or before) and then expanded in number and geographical reach, resulting in the polymorphism being in mulriple sequences in Mexico, as well as Washington (also multiple), Oregon, and New Zealand in April, followed by additional isolates in two locations in China as well as Argentina. Somehwere along the line, this backbone also picked up H274Y, but was not detected in any of the unselected isolates. It only appeared in two patients in Seattle after they had begun treatment, and as seen in the travel log, both samples from Washington had the defining polymorphism (A1231G). One had also acquired an additional polymorphism that was seen in a few other pandemic sequences, signaling recombination between a sequence with the original marker(A1231G), and H274Y and the rarer polymophism in the other sub-clade.

[HenryN]

Re: Sequences from Tamiflu Resistant Immunocompromised Patients Released

Well, then I'm confused with respect to the distinguishing polymorphisms of the Washington/29 isolate. The NA segment has S82P which set it apart from all other Swine Flu isolates except Florida/04 and 08, two isolates that were collected in late April. So the earlier polymorphism is S82P, and the H274Y change that caused Tamiflu resistance came later.

Or, are we saying that the H274Y has been circulating since mid-April?

Below is the travel log of the sequences on the same branch (they all have the same polymorphism, which is also in the latest Argentina sequence at GISAID (not released to Genbank). This polymorphism (A1231G) came FIRST.

gb|GQ499337.1| Influenza A virus (A/Washington/28/2009(H1N1))... 40.1 0.047
gb|GQ499335.1| Influenza A virus (A/Washington/29/2009(H1N1))... 40.1 0.047
gb|GQ465707.1| Influenza A virus (A/Canada-NS/RV1565/2009(H1N... 40.1 0.047
gb|GQ465699.1| Influenza A virus (A/Canada-NS/RV1554/2009(H1N... 40.1 0.047
gb|GQ465698.1| Influenza A virus (A/Canada-NS/RV1551/2009(H1N... 40.1 0.047
gb|CY044165.1| Influenza A virus (A/Mexico/48N/2009(H1N1)) se... 40.1 0.047
gb|GQ433898.1| Influenza A virus (A/Jiangsu/1/2009(H1N1)) seg... 40.1 0.047
gb|GQ377086.1| Influenza A virus (A/Oregon/07/2009(H1N1)) seg... 40.1 0.047
gb|GQ377074.1| Influenza A virus (A/Washington/18/2009(H1N1))... 40.1 0.047
gb|GQ377048.1| Influenza A virus (A/Washington/17/2009(H1N1))... 40.1 0.047
gb|GQ338393.1| Influenza A virus (A/Washington/08/2009(H1N1))... 40.1 0.047
gb|GQ338371.1| Influenza A virus (A/Washington/10/2009(H1N1))... 40.1 0.047
gb|GQ323562.1| Influenza A virus (A/Florida/10/2009(H1N1)) se... 40.1 0.047
gb|GQ323543.1| Influenza A virus (A/Washington/09/2009(H1N1))... 40.1 0.047
gb|GQ323507.1| Influenza A virus (A/Washington/14/2009(H1N1))... 40.1 0.047
gb|GQ223445.1| Influenza A virus (A/GuangzhouSB/01/2009(H1N1)... 40.1 0.047
gb|GQ221696.1| Influenza A virus (A/GuangzhouSB/01/2009(H1N1)... 40.1 0.047
gb|CY040890.1| Influenza A virus (A/Mexico/47N/2009(H1N1)) se... 40.1 0.047
gb|GQ150333.1| Influenza A virus (A/Christchurch/2/2009(H1N1)... 40.1 0.047
gb|GQ132158.1| Influenza A virus (A/Canada-NS/RV1536/2009(H1N... 40.1 0.047
gb|GQ132154.1| Influenza A virus (A/Canada-NS/RV1538/2009(H1N... 40.1 0.047
gb|GQ122098.1| Influenza A virus (A/Christchurch/2/2009(H1N1)... 40.1 0.047

[wotan]

Re: Sequences from Tamiflu Resistant Immunocompromised Patients Released

Well, then I'm confused with respect to the distinguishing polymorphisms of the Washington/29 isolate. The NA segment has S82P which set it apart from all other Swine Flu isolates except Florida/04 and 08, two isolates that were collected in late April. So the earlier polymorphism is S82P, and the H274Y change that caused Tamiflu resistance came later.

Or, are we saying that the H274Y has been circulating since mid-April?

Well, given that the outbreak started in the latter part of flu season, is it unreasonable to assume that a very small population may have acquired that trait as early as April?

[HenryN]

Re: Sequences from Tamiflu Resistant Immunocompromised Patients Released

Well, then I'm confused with respect to the distinguishing polymorphisms of the Washington/29 isolate. The NA segment has S82P which set it apart from all other Swine Flu isolates except Florida/04 and 08, two isolates that were collected in late April. So the earlier polymorphism is S82P, and the H274Y change that caused Tamiflu resistance came later.

Or, are we saying that the H274Y has been circulating since mid-April?

No. S82P isn't on the branch containing the Washington isolates (other than WA/59). It was added to Washington/29 LATER (after the formation of the Washington branch). S82P was apended onto the Washington backbone via recombination (which is also true fro H274Y). You might have to look at a phylogentic tree to understand what came first (on a given isolate, not the entire database).

The ordering is pretty straight forward. Those who want to ignore recombination would say that S82P on different branches is a coincindent random mutation (which becomes absurd fairly quickly, since the number of new additions is small and the "coincidences" happen over a short time frame over and over and over.....).

[Mamabird]

Re: Sequences from Tamiflu Resistant Immunocompromised Patients Released

when you have
--X--
--XY-
--XZ-

then X was probably earliest

Well, then I'm confused with respect to the distinguishing polymorphisms of the Washington/29 isolate. The NA segment has S82P which set it apart from all other Swine Flu isolates except Florida/04 and 08, two isolates that were collected in late April. So the earlier polymorphism is S82P, and the H274Y change that caused Tamiflu resistance came later.

Or, are we saying that the H274Y has been circulating since mid-April?