Tracking Mutations: Novel H1N1 Leading to Possible Increase in Virulence/Transmissi

oramar · Jun 17, 2009

I get the feeling that this has all happened before. In 1918 we got to see the effects of the changes in the virus. In 2009 we are seeing the actually changes which being documented as they occur. Fascinating and scary at the same time.

indigo girl · Jun 17, 2009

Back to the info from flutrackers, it looks like only that one Shanghai sequence is a problem so we cannot say that this is going to occur everywhere. It may not occur again, but it is very possible as this is a very common characteristic of all seasonal influenzas and as such it should be easily acquired by swine flu.

There are some changes that researchers will be looking for that will alert them to some differences in the way the virus will behave.

This link is to a thread that probably will be updated as changes are noticed so you might want to bookmark it, and check it periodically as more information becomes available.

Influenzas are very fast mutating viruses. We are going to see changes but no one can say for sure what they will be.

http://www.flutrackers.com/forum/showthread.php?t=110649

indigo girl · Jun 17, 2009

CDC rejects report of mutant H1N1 strain in Brazil

http://www.cidrap.umn.edu/cidrap/content/influenza/swineflu/news/jun1709flustrain.html

The Centers for Disease Control and Prevention (CDC) and other experts have rejected a report that a new strain of the novel H1N1 influenza virus has been identified in a Brazilian patient.
Scientists at Adolfo Lutz Bacteriological Institute in Sao Paolo said they found the new strain in a local patient who has recovered, according to a Medical News Today (MNT) report, which was based on information from the institute and Agence France-Presse.
The story said the scientists found "a number of discrete alterations in nucleotide and amino acid sequences" in the isolate's hemagglutinin (HA) gene. They also analyzed the matrix-protein (MP) gene and found no changes.
But CDC spokesman Joe Quimby in Atlanta discounted the report that the isolate is a new strain. "Our scientists have no knowledge of a new strain of novel A H1N1 influenza," he said.
"It's the same strain, it's not a new strain," Quimby added.

indigo girl · Jun 18, 2009

E627K Acquisition in Swine H1N1 Raises Pandemic Concerns

http://www.recombinomics.com/News/06180901/H1N1_Swine_E627K.html

I thought for sure that we would be hearing from Dr. Henry Niman about the recent acquistion of E627K in swine flu. I had been waiting for his commentary, and here it is. We must also give full credit to Mamabird over at flutrackers.com for her far sighted vigilance in seeking out this information. I believe that she was the first person anywhere to notice the change and to post about it publicly. Kudos!

It is possible that CDC or WHO was aware and had not said anything. At any rate, if they were not, they certainly are now since they do admit to reading the flu forums and blogs

You might have some difficulty understanding some of Dr. Niman's commentary. That's OK. You will understand enough of it to know why this information is of concern. Swine flu, as we all know is very transmissible unlike bird flu which has not yet picked up that ability.

The recently released PB2 sequence from a patient in Shanghai...contains E627K. This is the first reported acquisition of this change, which is present in virtually all human influenza A isolates, including the pandemic strain from 1918.
The current isolate, A/Shanghai/71T/2009, was collected May 31, 2009 and the sequence was deposited at Genbank on June 10, 2009. The isolate is closely related to the swine H1N1 currently spreading worldwide, ...
Acquisition of E627K is a concern because it allows for optimal replication at 33 C, the temperature of a human nose in the winter, in contrast to E627, which is in the avian version of PB2 and allows for optimal replication at 41 C, the body temperature of birds. The PB2 in the swine is of avian origin, and A/Shanghi/71T/2009 is the first public sequence from the circulating sub-clade with E627K.
In human seasonal flu, activity peaks in the winter months and only minimal levels are detected in humans over the summer. However, the swine flu has remained active and almost all influenza A detected in the northern hemisphere at this time is swine flu.
The appearance of E627K raises concerns that the level of swine flu with E627K will markedly increase in colder months. In 1918, the flu in the spring was mild, but the fall version of the virus, which had E627K, was much more virulent and targeted young, previously healthy adults, as previously reported for the outbreak in Mexico, and now being reported in United States and Canada, where levels are highest. This younger population target is also being reported in other countries worldwide.
Although the current case fatality rate is low, small changes like E627K could increase viral load, leading to high cytokine levels, which are associated with many of the deaths due to the current "milder" version of the swine H1N1. In contrast to the recent isolate from Brazil, A/Sao Paulo/1454/2009 which is identical to the HA consensus sequence, the A/Shanghi/71T/2009 has a unique change that is not found in closely related sequences.
This change is found in other H1N1 swine sequences (see list here) and the region surrounding E627K matches H3N2 seasonal flu in circulation in 1995. Moreover, the downstream region matches sequences found in human lethal infections in the H7N7 outbreak in the Netherlands in 2003, as well as the H5N1 outbreak in Hong Kong in 1997 (see matches here). However, in the above outbreaks, transmission to humans was not efficient, and the number of deaths was limited.
The only reported death in the H7N7 outbreak was a veterinarian infected with H7N7 containing E627K (and that case remains the only example of a human fatal bird flu infection not involving H5N1). The H5N1 sequence from Hong Kong was used to show the association of E627K with increased virulence in mice.
Thus, the acquisition of PB2 E627K in the swine H1N1 is readily explained by homologous recombination with locally related sequences containing E627K, which was a concern when the swine H1N1 begun transmitting efficiently in humans . This sustained efficient transmission lead to a phase 6 designation for the 2009 Pandemic.
The acquisition of E627K creates concern that the virus will evolve into a more lethal agent that will be associated with an increased case fatality rate in previously healthy young adults, as was seen in the 1918 Pandemic.

indigo girl · Jun 18, 2009

http://www.flutrackers.com/forum/showpost.php?p=250657&postcount=59

Another sequence with the same change noted over at flutrackers. They do good work between their translators, and the reading of as well as interpretation of what the viral seqences mean. They were also the first in the US to pick up on what was going on in Mexico as well, via translations, and relate it to the cases in the US before the CDC or the media even noticed! They are an amazing group of people. Can you tell how much I admire their work?

A second A/Shanghai/71T sequence has been posted to GISAID, this time with c2 passage. Same result. Lab error beginning to seem less likely. Lets continue to keep a lookout for other isolates with this nasty change.

indigo girl · Jun 23, 2009

More Commentary on PB2 E627K

http://www.recombinomics.com/News/06220901/H1N1_Buffalo.html

The clustering of two critically ill students raises concerns about the emergence of a more lethal strain of Pandemic H1N1.
The two serious cases may simply reflect the high level of H1N1 infections in the area.
However, the state agency and media reports do not describe any pre-existing conditions for either student and these two cases follow two announcements by the city of New York of 14 deaths in May. Seven were announced on Tuesday, and seven more were announced on Friday, but these announcements lacked detail. All had died in May and all were between the ages of 25-64. However, there was no additional information and no mention of pre-existing conditions.
The significance of the failure to mention pre-existing remains unclear. Many of the prior death were said to have pre-existing conditions, but there was little evidence pointing to a strong role of these conditions in the deaths. In Mexico, most of the fatalities were in young adults who did not have pre-existing conditions.
Recent comments in press conferences by WHO and the CDC have added to the uncertainty. At the WHO press release introductory remarks by Kieji Fukuda cited 249 deaths worldwide, which raised questions, since the official tally at the time was only 140 deaths. Last week at the CDC press conference, opening remarks by Dan Jerrnigan indicated that 40% of the fatal cases had pre-existing conditions, which was much lower than the 70% cited for the released cases. In both instances the press conference transcript was edited to reflect the numbers for the released cases. However, the delay in the acknowledgement of the cases in New York raises concerns that the higher number of fatal cases and the lower percentage of cases with pre-existing condition reflects the current number of confirmed cases, which includes a high percentage of fatal cases without pre-existing conditions.
The clustering of the critical cases in Buffalo raises concerns of a Pandemic strain that is evolving and becoming more lethal.
One such change that could increase lethality is PB2 E627K. This polymorphism is present in seasonal influenza A, including the 1918 pandemic strain. It allows for optimal replication at lower temperatures, which may lead to a more transmissible and lethal Pandemic strain. This change has been reported for an isolate from Shanghai from a patient (22F) who recovered. The collection date, location, age and gender, suggests that the isolate came from a Chinese national who was a student in the US and flew to Shanghai on a flight that originated in New York with a lay-over in Hong Kong. Thus, the virus could have originated in New York, Hong Kong, or Shanghai, since the student developed symptoms shortly after arriving in Shanghai. The sequencers in China promptly released full sequences on all eight gene segments, and sequenced a clone to confirm that the E627K was not a sequencing error. The sequence of the clone exactly matched the original sequence.
However, this change may not offer much advantage at this time of the year in the northern hemisphere, but the change could appear in the southern hemisphere or could signal a change that is more widespread and circulating in the Hong Kong area. In any event, the presence of E627K in pandemic H1N1 strongly suggests that it will emerge in the fall, which is cause for concern.
The Buffalo cluster however raises concerns of additional changes, so sequencing of the isolate from the fatally infected middle school student would be useful.

indigo girl · Jun 25, 2009

http://www.flutrackers.com/forum/showpost.php?p=254953&postcount=121

so, the cdc is aware of this change...

the cdc's view from the leaked june 19 cdc director's daily briefing -
http://www.astho.org/pubs/19jun20091...689c9b4cdb0ccd (slide 13)
quote:
isolate reported in china with genetic mutation possibly representing increased adaptation to humans.
-a/shanghai/71t/2009 sequence posted by shanghai public health clinical center (unknown reliability)
-change is due to mutation rather than reassortment with seasonal influenza since remainder of gene is similar to previous sequences
-no clinical data on associated case

well, they could just ask the chinese for the clinical data, couldn't they? they just might tell them.

indigo girl · Jun 26, 2009

http://www.flutrackers.com/forum/showpost.php?p=254197&postcount=1

Notes on a change that will make a difference in the cooler weather of our upcoming flu season.

As A/H1N1 flu continues to infect large numbers of people worldwide, flu and gene researchers study viral sequences (a sequence can be thought of as a viral “fingerprint”) to determine how changes in the sequence affect the ease of transmission and virulence.
Recently, a sample isolated from a case in Shanghai, China, showed a change in the segment that controls the optimal temperature for virus replication. Because of its avian components, the H1N1 virus has so far reproduced best at a temperature of 41 deg. C, or 106 deg. F., the temperature of a bird’s upper respiratory tract. The change in the Shanghai isolate would allow the virus to replicate best at the cooler temperature of human upper respiratory tracts, thus allowing for easier transmission by sneezing or coughing. The change has also been associated with a higher incidence of neurological complications, such as encephalitis, in other strains of influenza.
Scientists and researchers continue to monitor potential shifts in the H1N1 virus as it spreads around the world.

http://www.flutrackers.com/forum/showpost.php?p=255790&postcount=4

Flutrackers found this change before scientists made any announements of this possible problem. I want to give them full credit for this information.

indigo girl · Sep 29, 2009

E627K in the Netherlands

http://www.promedmail.org/pls/otn/f?p=2400:1001:2241587877719755: :no: :F2400_P1001_BACK_PAGE,F2400_P1001_PUB_MAIL_ID:1000,79432

We would like to report 2 patients in The Netherlands, diagnosed with
influenza pandemic A(H1N1) 2009 virus infection that had a mutation (E627K)
in the basic polymerase 2 (PB2) protein. This mutation has previously been
associated with increased efficiency of replication and possible virulence
changes in other influenza A viruses.
The investigation identified a specific geographic region in the north of
The Netherlands as the place where viruses with the same genetic background
have circulated between mid July and mid August [2009]. No other cases
carrying the PB2 mutation have been identified.
On 15 Sep 2009, the 1st influenza A(H1N1)v virus with a glutamic acid to
lysine mutation at position 627 (E627K) in PB2 was identified through
routine sequence analysis of clinical samples from a diabetic patient
infected with A(H1N1)v virus. The 1st day of illness was on 9 Aug 2009,
when the patient was vacationing on one of the West Frisian Islands in the
wetlands north of The Netherlands (Waddenzee). He had a relatively mild
course of illness. Subsequent retrospective tracing of geographically
linked A(H1N1)v cases from the national databases led to the identification
of 24 additional A(H1N1)v confirmed cases throughout the country that had
stayed on the same popular holiday island during July and August. Sequence
analysis of 12/24 clinical specimen available at the institute identified
10 A(H1N1)v viruses that clustered with the virus obtained from the
diabetic index patient based on unique mutations in the NA gene and PB2
gene. Only one of these had the PB2 E627K mutation. This virus was isolated
from a family contact of an adolescent girl who returned from a one-week
stay on the same island on Mon 20 Jul 2009 with high fever and coughing.
This girl had been camping with a group of 16 boys and 8 girls that shared
2 tents. Almost all members of this group reportedly had been ill, and
influenza A(H1N1)v infection had been diagnosed in 2 other persons
belonging to the same camp. The girl was ill for a week, with full recovery
after 2 weeks. Our 2nd case with a virus shedding carrying the PB2 mutation
is the younger sister and became ill on Thu 23 Jul 2009. She was treated
with oseltamivir and recovered fully after one week. Both parents remained
free from symptoms.
As the mutations were identified more than one month after initial
detection, no further contact investigations were done. Municipal health
services were informed about the local disease activity. Since 15 Aug 2009,
mild influenza cases are no longer notifiable in The Netherlands, so we
have no information on possible onward transmission. No clusters of illness
(for example, from schools) were reported in the health regions involved
(including the island), and surveillance data from a national
physician-based sentinel network showed low ILI activity for the
Netherlands. Samples from 22 patients hospitalized with influenza A(H1N1)
in July and August did not have the PB2 mutation.
PB2 627K is consistently found in human influenza A viruses, but rarely in
avian-derived viruses. The E627K mutation may result in enhanced virus
replication efficiency in humans, possibly by adjustment to host body
temperature or cellular cofactors, and has previously been shown to be
associated with fatal cases of HPAI H5N1 and H7N7 virus infection in
humans. Until now, A(H1N1)v viruses with Influenza pandemic (H1N1) 2009
(57): in PB2 have not been reported, and the clinical and epidemiological
relevance of our finding remains unclear.
Preliminary experiments in ferrets using reverse genetics-derived new
influenza A(H1N1)v viruses with the E267K mutation in PB2 did not indicate
increased shedding, virulence or transmissibility. Further experiments as
well as increased molecular surveillance to monitor the situation are ongoing.

(hat tip pfi/cactus)

indigo girl · Sep 30, 2009

silent spread of e627k in pandemic h1n1 patients

http://www.recombinomics.com/news/09290902/h1n1_e627k_ss.html

h274y is a genetic marker indicating that a strain of influenza is resistant to the antiviral drug, tamiflu.

e627k indicates that the virus is able to replicate efficiently at 33 c, the temperature of a human nose in the winter. e627 is in the avian version of pb2, and indicates replication at 41 c, the body temperature of birds.the appearance of e627k is a great concern as we move into the colder months of our winter season.

e627k is present in virtually all human influenza a isolates, so it is not surprising that this influenza should pick up this polymorphism as it adapts to human hosts. it has previously been shown to be associated with fatal cases of hpai h5n1 and h7n7 (bird flu) viral infections in humans.

sequence analysis of 12/24 clinical specimen available at the institute identified 10 a(h1n1)v viruses that clustered with the virus obtained from the diabetic index patient based on unique mutations in the na gene and pb2 gene. only one of these had the pb2 e627k mutation. this virus was isolated from a family contact of an adolescent girl who returned from a one-week stay on the same island on mon 20 jul 2009 with high fever and coughing.
the above description from a promed report on isolates in the netherland raise concerns that virus with e627k is silently transmitting. this polymorphism was found in two of thirteen similar sequences, which do not appear to be linked. the index case for the detection is not directly linked to the sibling described above. it is unclear if the adolescent girl described above had e627k. the failure to detect may have been due to a lack of a sample or the failure to isolate a virus or sequences with e627k from that patient.
however, it is unlikely that the two positive samples are due to independent spontaneous "mutations". it is much more likely that the e627k is in a mixture, and isolation procedures or sequencing approaches lead to frequent detection of the avian version of pb2, which has an e at pb2 position 627. the only prior report of e627k in a pandemic h1n1 sequence was from a patient in shanghai. the original sequence, as well as the first sub-clone had e627k, while a second sub-clone had e627.
this type of mixture could not only allow for the spread of e627k, but could also lead to elevated levels in the upper respiratory tract, due to the dominance of e627k, followed by increased levels of h1n1 in the lower respiratory tract due to a dominance of e627. the silent spread of e627k could be linked to the increased severity of pandemic h1n1 infections being reported at schools throughout the northern hemisphere.
like na h274y, the mixture produces sequences that are dependent on selection pressure. h274y has been most commonly detected in patients receiving oseltamivir, although at least two isolates were from patients not receiving oseltamivir, and the presence of h274y in the second camper in north carolina is probably not linked to oseltamivir use, since it has the same rare genetic marker found in the index case. similarly, the shared markers in the netherland isolates with e627k argue for transmission, but the two positives are not directly linked and most samples tested do not have detected e627k.
these data highlight the need for more aggressive cloning and sequencing. the acquistion of na h274y was predicted because if a high frequency in seasonal h1n1, while pb e627k is at high frequency in pb2 in seasonal h1n1 and h3n2. recombinations allows for the jump of both polymorphsism from a seasonal flu background to pandemic h1n1.
the presence of mixtures at pb2 position 627 could have catastrophic consequences, linked to site specific increases in viral load. similarly, release of sequences would allow for a fuller understanding of the dsitribution of the sub-clade isolated in the netherlands with and without e627k.

indigo girl · Sep 30, 2009

The Case of the (Possibly) Benign Mutation

http://afludiary.blogspot.com/2009/09/case-of-possibly-benign-mutation.html

Sometimes the influenza virus is real a mystery.
Scientists have been watching intently for months to detect any mutation in the H1N1 `swine’ flu virus that might add greater virulence, or perhaps antiviral resistance, to its repertoire.
While there are many possible mutations that can occur, the truth is we don’t know what a lot of those would mean for the `fitness’ of the virus.
One of the mutations scientists thought they understood reasonably well is the (E627K) substitution in the (PB2) protein; The swapping out of the amino acid Glutamic acid (E) at position 627 for Lysine (K).
Glutamic acid (E) at this position is a hallmark of avian influenza viruses, and is believed to make the virus better adapted to replicate at the higher temperatures commonly found in birds (41C).
Human flu viruses normally have Lysine (K) at position 627. That mutation supposedly makes the virus better adapted to replicate at the lower temperatures (roughly 33C) normally found in the upper human respiratory tract.
The H1N1 swine flu, which is a reassortment of human, swine, and avian viruses, carries this `avian’ style E627 marker.

Therefore, any change at that position from E to K (Glutamic Acid to Lysine) would be expected to improve human adaptability and potentially increase virulence and/or transmissibility of the virus.
Earlier this year we heard of an E627K mutation of the H1N1 swine flu virus detected in Shanghai (A/Shanghai/71T/2009, May 31st), which raised some eyebrows, but we’ve heard little else since then. The ProMed Mail report below doesn’t reference the Shanghai sample.
Now we get word of two virus samples from the Netherlands where this mutation has once again been observed.
But the good news, for now, is that ferret testing has shown no obvious increases in virulence or transmissibility. This comes as a genuine surprise to researchers, one of whom admits `he would have bet his car’ on this mutation increasing virulence.
This first report from The Canadian Press.
Dutch researchers find mutation linked to virulence in swine flu virus
(CP)
TORONTO — Dutch scientists are reporting they have found a key mutation in several swine flu viruses from the Netherlands, a mutation that in other flu viruses increases virulence.
But they say the mutation, on the PB2 gene, doesn't appear to cause more severe disease with the novel H1N1 virus.
The two people from whom the mutated viruses were isolated had standard cases of swine flu and when the mutated virus was tested in ferrets it didn't produce more severe disease.
(Continue . . .)
ProMed Mail carried an alert on this story last night, some of which is excerpted below:
INFLUENZA PANDEMIC (H1N1) 2009 (58): THE NETHERLANDS, PB2 MUTATION
******************************************************************
A ProMED-mail post

ProMED-mail is a program of the
International Society for Infectious Diseases

Date: Mon 28 Sep 2009
From: Marion Koopmans
We would like to report 2 patients in The Netherlands, diagnosed with influenza pandemic A(H1N1) 2009 virus infection that had a mutation (E627K) in the basic polymerase 2 (PB2) protein. This mutation has previously been associated with increased efficiency of replication and possible virulence changes in other influenza A viruses.
The investigation identified a specific geographic region in the north of The Netherlands as the place where viruses with the same genetic background have circulated between mid July and mid August [2009]. No other cases carrying the PB2 mutation have been identified.
PB2 627K is consistently found in human influenza A viruses, but rarely in avian-derived viruses. The E627K mutation may result in enhanced virus replication efficiency in humans, possibly by adjustment to host body temperature or cellular cofactors, and has previously been shown to be associated with fatal cases of HPAI H5N1 and H7N7 virus infection in humans.
Until now, A(H1N1)v viruses with Influenza pandemic (H1N1) 2009 (57): in PB2 have not been reported, and the clinical and epidemiological relevance of our finding remains unclear.
Preliminary experiments in ferrets using reverse genetics-derived new influenza A(H1N1)v viruses with the E267K mutation in PB2 did not indicate increased shedding, virulence or transmissibility. Further experiments as well as increased molecular surveillance to monitor the situation are ongoing.
As I said at the start of this, sometimes the influenza virus is a real mystery. We’ve watched and waited for this particular amino acid substitution with the expectation that it would herald a more human adapted virus.
Now that it has appeared, and no such behavioral changes have been observed, it suggests that there may be other factors – which we don’t understand – that must occur possibly in concert with this change to make the virus more virulent.
And so the mystery deepens.