Denmark Tamiflu Resistant Sequence Released

[HenryN]

Re: Denmark Tamiflu Resistant Sequence Released

For HA there are two polymorphisms in the 11F not in the 21F or 24M (the infection of the 11F was not linked to the 21F or 22M).

 

[HenryN]

Re: Denmark Tamiflu Resistant Sequence Released

For HA there are two polymorphisms in the 11F not in the 21F or 24M (the infection of the 11F was not linked to the 21F or 22M).

The HA sequences of 21F and 22M are identical supporting an infection of 21F by 22M. They both have the unusual (not in any other H1N1 pandemic sequence) HA travel log below (which has the Fort Dix polymorphism). The sequence below from Thailand was also from a patient infected with a swine H1N1, in 2005).

gb|CY040549.1| Influenza A virus (A/swine/North Carolina/4395... 32.2 11
gb|CY039991.1| Influenza A Virus (A/New Jersey/8/1976(H1N1)) ... 32.2 11
gb|FJ688266.1| Influenza A virus (A/swine/Thailand/HF6/2005(H... 32.2 11
dbj|AB434328.1| Influenza A virus (A/swine/Chachoengsao/NIAH5... 32.2 11
dbj|AB434320.1| Influenza A virus (A/swine/Chonburi/NIAH589/2... 32.2 11
dbj|AB434312.1| Influenza A virus (A/swine/Chonburi/NIAH977/2... 32.2 11
dbj|AB434304.1| Influenza A virus (A/swine/Chonburi/NIAH9469/... 32.2 11
dbj|AB434296.1| Influenza A virus (A/swine/Ratchaburi/NIAH550... 32.2 11
gb|EU798784.1| Influenza A virus (A/swine/Korea/Asan04/2006(H... 32.2 11
gb|EU798783.1| Influenza A virus (A/swine/Korea/JL04/2005(H1N... 32.2 11
gb|EU798781.1| Influenza A virus (A/swine/Korea/JL01/2005(H1N... 32.2 11
gb|EU798780.1| Influenza A virus (A/swine/Korea/Hongsong2/200... 32.2 11
gb|EU296605.1| Influenza A virus (A/swine/Chonburi/06CB2/2006... 32.2 11
gb|EU296603.1| Influenza A virus (A/swine/Chonburi/05CB1/2005... 32.2 11
gb|EU296601.1| Influenza A virus (A/swine/Chachoengsao/NIAH58... 32.2 11
gb|EU296599.1| Influenza A virus (A/swine/Chonburi/NIAH589/20... 32.2 11
gb|CY026427.1| Influenza A virus (A/swine/Jamesburg/1942(H1N1... 32.2 11
gb|CY022469.1| Influenza A virus (A/swine/Kansas/3228/1987(H1... 32.2 11
gb|EF101749.1| Influenza A virus (A/Thailand/271/2005(H1N1)) ... 32.2 11
gb|DQ431990.1| Influenza A virus (A/swine/Taiwan/CO935/2004(H... 32.2 11
gb|DQ447187.1| Influenza A virus (A/swine/Taiwan/CO935/2004(H... 32.2 11
gb|AY852271.1| Influenza A virus (A/swine/Guangdong/711/2001(... 32.2 11
gb|U80950.1|IAU80950 Influenza A virus hemagglutinin (HA) gen... 32.2 11
gb|U72666.1|IAU72666 Influenza A virus hemagglutinin (HA) gen... 32.2 11

 

[HenryN]

Re: Denmark Tamiflu Resistant Sequence Released

The HA polymorphism serves as a marker. It does not change the protein sequence of the virus, but it is unique to the two patients (21F and 22M).

 

[theforeigner]

Re: Denmark Tamiflu Resistant Sequence Released

I am still going through the sequences. The NA sequences for the other two isolates have no new changes, so the only difference between the three NA sequences is the H274Y (Tamiflu resistance) in the patient on prophylactic Tamiflu (confirming earlier media reports).

Thanks Niman and thanks for beeing so patient

 

[HenryN]

Re: Denmark Tamiflu Resistant Sequence Released

The PB1 travel log is the only newly acquired polymorphism, A178T, in the PB1 sequence. I suspect it is nonsynomous because it is a transversion (swaps a purine for a pyrimidine) but haven't looked yet. I believe the donor sequences is Brisbane/10 (but also haven't looked yet). The polymorphisms represents an "upgrade" so to speak (adding a human 2007 polymorphism to a 1993 sequence).

The A178T is in the PB2 sequence (not PB1 as stated above). The PB1 sequence has two polymorphisms. The one shared with the human H3N2 sequences from 2007 (A657G) is synonymous and there doesn't change the protein, yet is found in human H3N2 (from 2007, not 1993). The other polymorphism, T2200C, is non-synonymous and creates F730S. However, I did not find this polymorphism in the database.

Somewhat surprisingly, all THREE PB1 sequences from Denmark are IDENTICAL, suggesting these two PB1 markers identify a Denmark specific sequence. Thus, even though the HA sequences are distinct, all three have matching PB1 markers, that among pandemic H1N1 sequences, are ONLY in Denmark.

 

[AlaskaDenise]

Re: Denmark Tamiflu Resistant Sequence Released

Is it safe to say that all this represents a virus becoming more adapted to humans, thereby improving its ability to cause disease?

.

 

[HenryN]

Re: Denmark Tamiflu Resistant Sequence Released

Is it safe to say that all this represents a virus becoming more adapted to humans, thereby improving its ability to cause disease?

.

It is safe to say that these three have picked up a novel set of (Denmark) markers in PB1. Media reports suggest the 21F and 22M are contacts, so matches there are not surprising. However, the match with the 11F is a surprise, based on the HA sequence and indicates the virus is picking up regional markers, as was seen in Argentina, where all 11 isolates had a novel marker in PA.

The virus is mixing and matching and becoming more diverse. Travel between the areas with regional markers will increase diversity and increase evolution.

This virus is getting ready to rock and roll in the fall.

 

[AlaskaDenise]

Re: Denmark Tamiflu Resistant Sequence Released

It is safe to say.....
The virus is mixing and matching and becoming more diverse. Travel between the areas with regional markers will increase diversity and increase evolution.

This virus is getting ready to rock and roll in the fall.

At what point might this increasing diversity have implications for the effectiveness of vaccines?

.

 

[HenryN]

Re: Denmark Tamiflu Resistant Sequence Released

At what point might this increasing diversity have implications for the effectiveness of vaccines?

.

Non-synonymous changes in HA and NA will have the most impact on vaccines. When they start mixing and matching (recombining), they can quickly evolve away from the vaccine target.

 

[AlaskaDenise]

Re: Denmark Tamiflu Resistant Sequence Released

.....The other polymorphism, T2200C, is non-synonymous and creates F730S. However, I did not find this polymorphism in the database...........

Where did this never-previously-sequenced mutation (that assists the virus to evolve away from vaccines) come from?

.

 

[HenryN]

Re: Denmark Tamiflu Resistant Sequence Released

Where did this never-previously-sequenced mutation (that assists the virus to evolve away from vaccines) come from?

.

A sequence not in the database (the swine database is not very robust).

 

[Zac]

Re: Denmark Tamiflu Resistant Sequence Released

With the tamiflu resistance now becoming evident in swine flu, would it be better to keep relenza on hand rather than tamiflu? (in terms of which is more likely to remain effective around October)

 

[HenryN]

Re: Denmark Tamiflu Resistant Sequence Released

PB2 travel log

gb|GQ160588.1| Influenza A virus (A/Virginia/04/2009(H1N1)) s... 38.2 0.19

All three sequences have the above travel log (A178T).

 

[HenryN]

Re: Denmark Tamiflu Resistant Sequence Released

PB2 travel log
A/Denmark/523/2009
gb|CY040915.1| Influenza A virus (A/duck/Laos/P0117/2007(H5N1... 42.1 0.018

gb|AF342824.1| Influenza A virus (A/Wisconsin/10/98 (H1N1)) P... 34.2 2.9

 

[AlaskaDenise]

Re: Denmark Tamiflu Resistant Sequence Released

A sequence not in the database (the swine database is not very robust).

I hope this isn't using up my daily question allowance, but since you brought up the high portion of the PB1.....

Does swine flu have a truncated or intact PB1-F2? I ask after reading the following at the reference cited:

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 10, October 2006 1607
Influenza A Virus
PB1-F2 Gene

To the Editor: Recently, Chen and
co-workers described the expression
of an 11th influenza A virus protein,
designated PB1-F2 because this protein
is encoded in the +1 open reading
frame of the segment-2 RNA (1).

Later, Chen et al. presented a preliminary
analysis of 336 PB1 sequences
from GenBank (2). We have extended
the work on PB1-F2 and analyzed
1,864 partial and complete segment-2
sequences deposited in GenBank;
these sequences belong to 79 influenza
A virus subtypes. In summary, the
following 8 observations should
receive attention:
First, the size of PB1-F2 polypeptides
ranges from 79 to 101 amino
acids (aa); most isolates encode versions
of either 87 or 90 aa. Because
polypeptides of 79 aa are located
within mitochondria, their truncation
has no effect on the protein function.
The frequency of the 79-aa PB1-F2 is

≈5%.
Second, a functional PB1-F2 is
expressed by 92% of all segment-2
sequences, i.e., a polypeptide >78 aa.

The proportion of intact PB1-F2
varies according to host (humans
90%, swine 76%, other mammals
100%, birds 95%).

Third, the H1N1 subtype comprises
3 genetic lineages. One clade has 2
branches: 1 branch includes the
human viruses, with the pandemic
1918 virus at its root; the other branch
includes the classic swine viruses.

The third clade represents the
European porcine isolates. Although
all classic swine sequences have a
truncated PB1-F2 (in-frame stop
codons after 11, 24, and 35 codons),
the early human isolates (H1N1
sequences from 1918 through 1947)
have an intact PB1-F2. After 1956,
however, a mutation became prevalent
such that the recent sequences
starting from A/Beijing/1/56 terminate
after 57 codons. An exception to
this rule is A/Taiwan/3355/97. Two
human H1N1 isolates with an intact
PB1-F2 coding sequence cluster in
the H3N2 clade (A/Kiev/59/79,
A/Wisconsin/10/98). The PB1
sequences of European porcine
influenza A virus isolates cluster with
European porcine H3N2 and H1N2.

Fourth, all H2N2 sequences are
monophyletic and encode an intact
PB1-F2. Fifth, the main sequence
cluster of the H3N2 subtype comprises
3 branches: 1) porcine H3N2 and
porcine H1N2 sequences from the
United States, 2) porcine H3N2 isolates
from Hong Kong and human
H1N2, and 3) recent human H3N2
and some Japanese H3N2 isolates.
Most of these sequences encode an
intact PB1-F2.

Sixth, the cluster of European
porcine influenza A virus isolates
comprises the subtypes H1N1, H1N2,
and H3N2. The lack of distinct clades
for each subtype indicates frequent
reassortment in the evolution of these
viruses. Of the segment-2 sequences,
56% encode an intact PB1-F2.

Seventh, other porcine isolates of
various subtypes represent transspecies
infections or single reassortment
events. And eighth, the segment-
2 sequences of many avian
influenza A virus isolates encode
intact PB1-F2. Considerable proportions
of truncated PB1-F2 genes were
found in the H5N2, H6N6, H9N2,
and H13N2 subtypes. However,
because of the small number of
sequences available, this observation
may not be important.

In conclusion, PB1-F2 is
expressed in most avian and many
porcine influenza A virus isolates.
This finding contrasts with those in
the initial publication, which stated
that PB1-F2 is not expressed in many
animal isolates, particularly those of
porcine origin (1). Because PB1-F2
was described as a proapoptotic protein
probably counteracting the host
immune response, why numerous
human and porcine isolates lack this
protein without selective disadvantage
remains unclear.

Roland Zell,* Andi Krumbholz,*
and Peter Wutzler*

*Friedrich Schiller University, Jena,
Germany

References

1. Chen W, Calvo PA, Malide D, Gibbs J,
Schubert U, Bacik I, et al. A novel influenza
A virus mitochondrial protein that
induces cell death. Nat Med.
2001;7:1306–12.
2. Chen GW, Yang CC, Tsao KC, Huang CG,
Lee LA, Yang WZ, et al. Influenza A virus
PB1-F2 gene in recent Taiwanese isolates.
Emerg Infect Dis. 2004;10:630–6.
Address for correspondence: Roland Zell,
Institute of Virology and Antiviral Therapy,
Medical Centre at the Friedrich Schiller
University, Hans Knoell Str 2, D-07745 Jena,
Germany; email: Roland.Zell@med.uni-jena.de
　

In response: Zell et al. (1) performed
an extensive genetic investigation
of PB1-F2, based on up-to-date
GenBank sequences. Their sample
size (1,864) greatly outnumbered ours
(336) in a previous study (2) and thus
definitely better portrays the genetic
characteristics of PB1-F2. We appreciate
their analyzing these samples by
subdividing nonhuman strains into
different species, which we did not do
(2). Their analysis is especially meaningful
for the global pandemic threat
from avian influenza viruses, which
increases the need to study interspecies
adaptation and transmission.

Zell et al. found that 92% of PB1
RNA encodes a functional PB1-F2,
compared with our 79% (264/334),
which supports the increasingly crucial
role of PB1-F2 in influenza virology.
They found the proportion of
intact human PB1-F2 to be 90%, a
substantial boost from our 68%
(67/99), which was based on data
from late 2003 (2). This increase is
apparently caused by the increasing
number of human H3N2 sequences
(mostly encoding an intact PB1-F2
compared with H1N1) deposited in
the past 2 years.

Human H1N1 from 1918 through
1947 contains full-length PB1-F2,
whereas human H1N1 beginning in
1956 has a truncated PB1-F2 after
codon 57. As reported by Zell et al.,
only 3 human H1N1 strains contain
full-length PB1-F2: A/Kiev/59/79,
A/Taiwan/3355/97, and A/Wisconsin/
10/98. The PB1 genes of A/Kiev/
59/79 and A/Wisconson/10/98 were
found clustered with human H3N2 as
a result of natural reassortment
between human H1N1 and H3N2
strains. On the other hand, the asynonymous
mutation found on
A/Taiwan/3355/97 enabled the translation
to get past the usual stop codon
at position 58, which other H1N1
strains exhibit. A/Taiwan/3355/97
(H1N1) was isolated from a patient
with severe pneumonia. Animal study
has demonstrated that the existence of
full-length PB1-F2 contributed to
pathogenesis in mice (3). We speculate
that the expression of a full-length
PB1-F2 may contribute to disease
severity in humans.

The C-terminal domain of PB1-F2
contains the mitochondrial signal and
can trigger apoptosis in specific
immune-related cells. Our recent
work (4) comparing avian and human
influenza A viruses also found that
many species-associated amino acid
signatures are located on the C terminal
of PB1-F2. This finding highlights
the importance of further investigating
the role of PB1-F2 on interspecies
infection.

Guang-Wu Chen*
and Shin-Ru Shih*

*Chang Gung University, Taoyuan, Taiwan

References

1. Zell R, Krumbholz A, Wulzler P. Influenza
A virus PB1–F2 gene [letter].Emerg Infect
Dis. 2006;12:1607–8.
2. Chen GW, Yang CC, Tsao KC, Huang CG,
Lee LA, Yang WZ, et al. Influenza A virus
PB1–F2 gene in recent Taiwanese isolates.
Emerg Infect Dis. 2004;10:630–6.
3. Zamarin D, Ortigoza MB, Palese P.
Influenza A virus PB1–F2 protein contributes
to viral pathogenesis in mice. J
Virol. 2006;80:7976–83.
4. Chen GW, Chang SC, Mok CK, Lo YL,
Kung YN, Huang JH, et al. Genomic signatures
of human versus avian influenza A
viruses. Emerg Infect Dis 2006:9:1353–60.
Address for correspondence: Guang-Wu Chen,
Chang Gung University, Department of
Computer Science and Information
Engineering, 259 Wen-Hua 1st Rd, Kwei-shan ,
Taoyuan, Taiwan 333; email: gwchen@
mail.cgu.edu.tw　

http://www.cdc.gov/ncidod/EID/vol12no10/pdfs/06-0511_06-0909.pdf

 

[HenryN]

Re: Denmark Tamiflu Resistant Sequence Released

With the tamiflu resistance now becoming evident in swine flu, would it be better to keep relenza on hand rather than tamiflu? (in terms of which is more likely to remain effective around October)

Both would be best (Relenza resistance may also be silently circulating and may appear as Relenza use increases).

 

[gsgs]

Re: Denmark Tamiflu Resistant Sequence Released

no PB1-F2 in **

but it is in related classical swine H1N1, so may be acquired
by reassortment from other swine

what's with N40 (new 12 th protein) I haven't yet checked

 

[gsgs]

Re: Denmark Tamiflu Resistant Sequence Released

                                    002 02  0001 00011 000 00 000 
                                    141 61  6674 24612 378 46 233 
                                    766 48  5770 94244 142 90 756 
                                    743 59  8878 87438 623 20 957 
-codon-position--------------------- 2   2  1  1 1     111     11 
---Index----------------------------AGG AT  TGCC GGGGG GAC GG GTA 
  1 >A Mexflu/index/2009/02/01      ... ..  .... ..... ... .. ... 
  2 >M Cancun/Index/2009/04/15      ..A ..  A..T A..AA AG. AA ..G 
  3 >A/Denmark/523/2009/06/04,11f   TAA GC  AT.T AAAAA AG. AA A.G 
  4 >A/Denmark/524/2009/06/04,22m   T.A GC  A.TT A.AAA AG. AA ACG 
  5 >A/Denmark/528/2009/06/09/21f   T.A GC  A.TT A.AAA AGT AA ACG

 

[Mamabird]

Re: Denmark Tamiflu Resistant Sequence Released

"I hope this isn't using up my daily question allowance, but since you brought up the high portion of the PB1.....

Does swine flu have a truncated or intact PB1-F2? I ask after reading the following at the reference cited:"

AD: PB1 of Swine Flu has a stop condon (taa) that prevents the formation of the open frame 2 protein. Wouldn't take much of a change to allow formation, and the F2 characterics of Swine Flu appear to be problematic, closely matching prior pandemic viruses. Bears close monitoring.

 

[AlaskaDenise]

Re: Denmark Tamiflu Resistant Sequence Released

.......Does swine flu have a truncated or intact PB1-F2? I ask after reading the following at the reference cited:"

AD: PB1 of Swine Flu has a stop condon (taa) that prevents the formation of the open frame 2 protein. Wouldn't take much of a change to allow formation, and the F2 characterics of Swine Flu appear to be problematic, closely matching prior pandemic viruses. Bears close monitoring.

thanks.

That issue looked like something to think about.

The more I read, the more I am amazed at how well this virus can perform its goal over many years by recycling strategies.

.