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Commentary
D225G/N
Emergence In H1N1
Variants
Recombinomics
Commentary 01:38
October 22, 2010
We show that N142D is centrally located in this epitope.
HA D111N is almost exclusively found in combination with another mutation, HA V267A, which is located at an internal beta sheet below the receptor binding pocket facing the Sa epitope (Figure 2). Exchanging valine for the smaller alanine at this position creates a small cavity which may slightly alter the surface of the epitope on top and could add to the effects of N142D.
While genetic differences were apparent in this variant group of pandemic influenza A(H1N1) viruses, when they were assessed for antigenic variation in haemagglutination inhibition assays (HI) using ferret antisera raised to A/California/7/2009-like viruses and viruses from the new variant group (e.g. A/Singapore/548/2010, A/Brisbane10/2010), no differences in titres were apparent, indicating that these viruses were not antigenically distinguishable from the reference and vaccine virus A/California/7/2009 (Table 2).
The recently released report on an emerging H1N1 variants noted that the virus could infect and kill patients who had previously been vaccinated with pandemic H1N1 raising concerns that the emerging strain could spread among those who were infected by or vaccinated against pandemic H1N1. The above comments describe three non-synonymous changes in HA (D95N, N126D, and V251A using H3 numbering) which could contribute to vaccine failure, but the new strain was similar to the vaccine target, A/California/7/2009 when used in HI assays, as seen in Table 2.
However, Table 2 includes pandemic H1N1 as well as seasonal H1N1 and the ferret antisera fail to distinguish (only a two fold difference in titer is observed) between the two very different targets, signaling a lack of sensitivity in the assay.
CORRECTION: Table 2 does not contain seasonal H1N1 targets. A/BRISBANE/2013/2009 is a pandemic H1N1 isolate. The July 1, 2009 collection date listed as 1/7/2009 was mistakenly read as January 7, 2009 leading to the assumption that it was a seasonal H1N1 isolate collect prior to the earliest pandemic H1N1 collection date. Similarly, A/Illinois/9/2007 was assumed to be seasonal H1N1 because of the collection date, but is in fact a swine sequence isolated from a human in 2007. Although this sequence does not appear to be public, it is likely to be similar to other swine sequences in humans in 2007 like A/Ohio/1/2007, which is swine, but easily distinguished from pandemic H1N1. Thus, although all targets in Table 2 have a swine origin, the 2 fold or less titer difference between A/Illinois/9/2007 and all isolates except A/Lviv/N6/2009 highlights the lack of sensitivity in the assay and the low probability of identifying vaccine failure in the pandemic H1N1 variant.
The use of ferret antisera to detect low reactors in H1N1 has been controversial, and lab to lab variation has been significant. It is noteworthy that the only target in Table 2 to show significantly reduced titers against pandemic as well as seasonal H1N1 is A/Lviv/N6/2009, which was originally sequenced from an Oropharyngeal sample from a deceased patient (26M) in Ukraine by Mill Hill. The sequence has D225G and was designated a low reactor by Mill Hill. The CDC subsequently sequenced an isolate by the same name which also had D225G as well as G158E. However, the CDC did not designate the isolate with G158E and D225G a low reactor, even though many labs, including the CDC, had designated other isolates with G158E as low reactors and Mill Hill had designated Lviv/N6, a low reactor. It is likely that the A/Lviv/N6/2009 used in Table 2 had D225G and possibly G158E and was clearly a low reactor. The ferret antisera against California/7 produced a titer of 640 when tested against a California/7 target, but the titer was reduced 8 fold when tested against Lviv/N6, supporting prior results by Mill Hill, which designated isolates with D225G or G158E as low reactors.
Initial sequences from Australia had D225G as well as D225N and both polymorphisms had been found in autopsy lung tissue from patients in Ukraine as well as Russia. Some sequences from Russia which had D225G also had G158E. Russian isolates with D225G and/or G158E were generally grown in eggs, although the autopsy lung sequences in Russia and Ukraine were direct sequences from the clinical samples.
Detection of D225G and G158E can be technically challenging because both polymorphisms are frequently found as pseudo-species involving wild type, D225G and D225N and detection is enhanced through the use of chicken egg embryos because of selection in cells which have gal 2,3, which is largely lacking in mammalian cell lines such as MDCK.
The early Oceana variant sequence with D225G was isolated in eggs as were all three sequences (from the same patient) with D225N.
Thus, the vaccine failures seen in Australia and New Zealand may have been due in part to D225G and/or D225GN which were not widely detected, but may have been selected against in samples from the upper respiratory tract, or virus isolated on mammalian cells lacking gal 2,3 receptors.
The designation of the Ukraine isolate with D225G as a low reactor raised concerns that D225G frequencies could be increased in emerging variants, and the vaccine failures in patients fatally infected with the recently described variant increase pandemic concerns.
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