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Published Date: 2010-04-09 13:00:03
Subject: PRO> Influenza pandemic (H1N1) (28): Hong Kong SAR, Norway, D222G mutation
Archive Number: 20100409.1147

INFLUENZA PANDEMIC (H1N1) (29): HONG KONG SPECIAL ADMINISTRATIVE
REGION, NORWAY, D222G MUTATION
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[1] Hong Kong SAR
[2] Norway

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[1] Hong Kong SAR
Date: Thu 8 Apr 2010
Source: Eurosurveillance, Volume 15, Issue 14 [edited]
<http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19534>

Letter: Association of D222G substitution in haemagglutinin of 2009
pandemic influenza A (H1N1) with severe disease
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The preferential binding of influenza virus to sialic acid-alpha2,3-
galactose (alpha2,3 receptor) or sialic acid-alpha2,6-galactose
(alpha2,6 receptors) may determine its tropism as alpha2,3 and
alpha2,6 receptors are dominant on lower and upper respiratory cells
respectively [1]. The recent glycan microarray analysis suggested
that the haemagglutinin (HA) D222G substitution could cause a shift
from alpha2,6 receptors to the mixed alpha2,3/alpha2,6 receptors
specificity which might increase binding to alpha2,3 receptors [2]
and contribute to severity of disease. This substitution in the HA
gene has been reported in samples of viruses obtained from cases with
mild to severe illness from around 20 countries, areas and
territories [3]. A recent study from Norway has evaluated the
clinical relevance of this substitution with severe and mild cases [4].

In an attempt to understand the relevance of HA D222G substitution
among pandemic influenza A (H1N1) causing infections in Hong Kong, HA
gene sequences from respiratory specimens and virus isolates of
severe and non-severe cases were examined. Cases were individuals who
had laboratory confirmed pandemic H1N1 influenza virus by either
viral culture or reverse transcription PCR (RT-PCR) of respiratory
specimens [5]. The severe cases were individuals classified by the
attending physician as being in a serious or critical condition.

From 1 May 2009 to 31 Jan 2010, 458 respiratory samples were
examined. Of 219 severe cases, nine (4.1 percent) showed D222G
substitution while none of the 239 non-severe cases showed D222G
substitution. Four of the nine cases died. The association of D222G
with severe disease was statistically significant (p=0.002, Fisher's
exact test, doubled one-sided). Other substitutions, of D222N (severe
cases, n=3; non-severe cases, n=1) and D222E (only in non-severe
cases, n=4) were also found. The 1st severe case appeared on 6 July
2009 and D222G substitution was detected in July, September, November
and December of the same year (These data are tabulated in the
original publication, to which interest readers are referred).

No distinct phylogenetic clusterings of the severe cases with D222G
substitution have been observed (data not shown). To put this in
perspective, from July 2009 to January 2010, the accumulated severe
cases were 244 while the number of isolates in our laboratory was 25
625. Priority of analysis has been given to severe cases over
non-severe cases, with 90 percent and 1 percent of cases analysed respectively.

Influenza is an RNA virus which evolves rapidly, frequently changing
surface structures. A recent study at the United States (US) Centers
for Disease Control and Prevention (CDC) reported 14 cases with D222G
substitution found only in virus isolates but not in the original
clinical specimens [3]. We observed similar finding with one
non-severe case showing D222G substitution in a virus isolate but not
in the original clinical specimen, however, for the other 9 severe
cases, we detected D222G substitution in both the virus isolate and
original specimen. Similar to the Norwegian study, we also found
mixed 222G and 222D in some severe cases [4]. Although experiments
with ferrets did not support a causal link of D222G substitution with
virulence [3], further study is warranted to elucidate the intriguing
relationship between D222G substitution and severe disease.

References
----------
(1) Shinya K, Ebina M, Yamada S, Ono M, Kasai N, Kawaoka Y. Avian
flu: influenza virus receptors in the human airway. Nature.
2006;440(7083): 435-6.
(2) Stevens J, Blixt O, Glaser L, Taubenberger JK, Palese P, Paulson
JC, et al. Glycan microarray analysis of the hemagglutinins from
modern and pandemic influenza viruses reveals different receptor
specificities. J Mol Biol. 2006;355(5):1143-55.
(3) World Health Organization. Preliminary review of D222G amino acid
substitution in the haemagglutinin of pandemic influenza A(H1N1) 2009
viruses. Wkly Epidemiol Rec. 2010;85(4):21-2 [English, French].
(4) Kilander A, Rykkvin R, Dudman S, Hungnes O. Observed association
between the HA1 mutation D222G in the 2009 pandemic influenza A(H1N1)
virus and severe clinical outcome, Norway 2009-2010. Euro Surveill.
2010;15(9) pii=19498. Available from:
<http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19498>
(5) Cheng PK, Wong KK, Mak GC, Wong AH, Ng AY, Chow SY, et al.
Performance of laboratory diagnostics for the detection of influenza
A(H1N1)v virus as correlated with the time after symptom onset and
viral load. J Clin Virol. 2010;47(2):180-5.

[Reported by: G C Mak1, K W Au1, L S Tai1, K C Chuang1, K C Cheng1, T
C Shiu1, W Lim 1
At: 1 Centre for Health Protection, Department of Health, Hong Kong SAR]

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[2] Norway
Date: Thu 8 Apr 2010
Source: Eurosurveillance, Volume 15, Issue 14 [edited]
<http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19535>

Reply: Association of D222G substitution in haemagglutinin of 2009
pandemic influenza A (H1N1) with severe disease
------------------------------------------------------------------------------------------------------------------
We appreciate the response to our paper [see ProMED-mail references
below] made by Dr. Mak and colleagues, whose data from Hong Kong SAR
appear to be in good agreement with what we have seen in Norway.

In our original study, we observed a significantly higher frequency
of D222G in patients with severe outcomes (including fatal) compared
to patients with mild disease. In fact, in both our data set and the
Hong Kong data, mutant viruses were not found among several hundred
mild cases. Furthermore, as can be seen from our published data, the
frequency may be higher also in fatal outcomes (8 of 27 cases) versus
severe non-fatal outcomes (3 of 34 cases). Comparing these
frequencies results in p=0.078 with Fisher's exact test (2-sided) and
p=0.046 with the Mid-P Exact test (2-sided). It would be interesting
to know if the new data from Hong Kong SAR can corroborate this
observation. Mak et al. report 4 fatal D222G cases and 5 non-fatal
severe D222G cases, but one would also need to know the total number
of fatal cases versus non-fatal severe cases analysed to make the
comparison. Hopefully, this information can be obtained.

D222G substitution in virus isolates only and not in the original
clinical specimens was found in one case in Hong Kong and 14 cases
reported by the United States (US) Centers for Disease Control and
Prevention [1]. We have also seen this virus culture artifact in one
case with mild disease. This case was counted as wild type in our
data set. This further underscores the importance to perform the
sequence analysis of the primary specimen.

The frequency of D222G mutant viruses in the severe cases is somewhat
lower in the Hong Kong data, compared to ours (4.1 percent in Hong
Kong data versus 18 percent in our data set). Whilst this difference
may represent a real variation in frequency, it may also arise from a
different composition of cases; e.g., if the proportion of fatal
cases were higher in the Norwegian sample of severe plus fatal cases.
Mak and colleagues also observed, as we did, that the 222G mutant
sometimes occurs in a mixture with non-mutated 222D genomes.

Sensitivity of detection of mutant viral genomes when occurring as
the minority variant in such mixtures may thus also influence the
observed frequency. We have been using a pyrosequencing assay to
identify the 222 genotype. Under ideal conditions this methodology
can reliably detect and quantitate a mutant when present in the total
virus population at levels as low as 10 percent [2]. However, in our
data set the initial finding of D222G mutants by pyrosequencing could
uniformly be verified by conventional sequencing. Therefore, since
the Hong Kong data come from a study focusing on this particular
position we assume that the methodology difference is not likely to
have caused the different frequencies in the 2 data sets. In the
overall global data, however, it is possible that some cases with
mutant/wild type mixtures have been overlooked and only the majority
sequence recorded.

References
----------
[1] World Health Organization. Preliminary review of D222G amino acid
substitution in the haemagglutinin of pandemic influenza A(H1N1) 2009
viruses. Wkly Epidemiol Rec. 2010;85(4):21-2. [English, French].
[2] Lackenby A, Democratis J, Siqueira MM, Zambon MC. Rapid
quantitation of neuraminidase inhibitor drug resistance in influenza
virus quasispecies. Antivir Ther. 2008;13(6):809-20.

[Reported by: A Kilander1, R Rykkvin1, S G Dudman1, O Hungnes 1
At: Department of Virology
Norwegian Institute of Public Health
Oslo, Norway]

--
Communicated by;
ProMED-mail
<promed@promedmail.org>

[These data from the investigations in Hong Kong SAR and Norway are
in agreement in that there is a good (but not absolute) correlation
between the presence of the D222G substitution in the HA protein and
disease severity. It is suggested that this mutation may affect
disease severity by causing a shift in virus binding from alpha2,6
receptors to mixed alpha2,3/alpha2,6 receptors in the respiratory
tract. These are interesting observations. However, it has been
observed also in these studies, and independently, that the D222G
substitution may be present also in virus isolates (i.e., virus
propagated in culture) only and not detectable in the original
clinical specimens. Perhaps the H222G mutation occurs at high
frequency and is favored in rapidly replicating virus either in
culture or in severely affected patients? - Mod.CP]

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