Frequency of D222G and Q223R Hemagglutinin Mutants of Pandemic (H1N1) 2009 Influenza Virus in Japan between 2009 and 2010

[tetano]

PLoS One. 2012;7(2):e30946. Epub 2012 Feb 17.
Frequency of D222G and Q223R Hemagglutinin Mutants of Pandemic (H1N1) 2009 Influenza Virus in Japan between 2009 and 2010.
Yasugi M, Nakamura S, Daidoji T, Kawashita N, Ramadhany R, Yang CS, Yasunaga T, Iida T, Horii T, Ikuta K, Takahashi K, Nakaya T.
Source

International Research Center for Infectious Diseases, Research Institute for Microbial Diseases (RIMD), Osaka University, Suita, Osaka, Japan.
Abstract
BACKGROUND:

In April 2009, a novel swine-derived influenza A virus (H1N1pdm) emerged and rapidly spread around the world, including Japan. It has been suggested that the virus can bind to both 2,3- and 2,6-linked sialic acid receptors in infected mammals, in contrast to contemporary seasonal H1N1 viruses, which have a predilection for 2,6-linked sialic acid.
METHODS/RESULTS:

To elucidate the existence and transmissibility of α2,3 sialic acid-specific viruses in H1N1pdm, amino acid substitutions within viral hemagglutinin molecules were investigated, especially D187E, D222G, and Q223R, which are related to a shift from human to avian receptor specificity. Samples from individuals infected during the first and second waves of the outbreak in Japan were examined using a high-throughput sequencing approach. In May 2009, three specimens from mild cases showed D222G and/or Q223R substitutions in a minor subpopulation of viruses infecting these individuals. However, the substitutions almost disappeared in the samples from five mild cases in December 2010. The D187E substitution was not widespread in specimens, even in May 2009.
CONCLUSIONS:

These results suggest that α2,3 sialic acid-specific viruses, including G222 and R223, existed in humans as a minor population in the early phase of the pandemic, and that D222 and Q223 became more dominant through human-to-human transmission during the first and second waves of the epidemic. These results are consistent with the low substitution rates identified in seasonal H1N1 viruses in 2008.

PMID:
22363521
[PubMed - in process]
PMCID:
PMC3281909

Free full text

http://www.ncbi.nlm.nih.gov/pubmed/22363521

 

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Re: Frequency of D222G and Q223R Hemagglutinin Mutants of Pandemic (H1N1) 2009 Influenza Virus in Japan between 2009 and 2010

From the full text study at PLoS:

Discussion:
This study used a high-throughput sequencing approach to analyze more than 2,000 clones from each of eight H1N1pdm samples obtained in 2009?2010. The results showed that α2,3 sialic acid-specific viruses containing D222G and/or Q223R substitutions within the HA molecule were present in the upper respiratory tract as a minor population in patients with mild H1N1pdm infections in the early phase (May 2009) of the pandemic in Japan. However, these substitutions nearly disappeared in the samples from five mild cases in Dec 2010, suggesting that the D222G and/or Q223R mutants showed low rates of human-to-human transmission. Thus, the newly emergent influenza A viruses may have been dual specific but not exclusively α2,6-sialic acid-specific during the early phase of the pandemic and adapted during multiple cycles of human-to-human transmission. These results are consistent with previous reports showing that, during previous pandemics, the receptor binding properties of avian influenza viruses changed after introduction into mammals [8]. In particular, positions 187 and 222 within the HA of H1 viruses and position 223 in H2 and H3 strains are critical for receptor binding specificity [8]. In the present study, the D187E substitution was not striking, suggesting that E187 had already disappeared in humans, or the original swine lineages, by May 2009. The N125D mutation, however, was detected (0.66?2.15%), even in the second wave of the outbreak (Table 2). However, as far as we know, there is no report indicating that this mutation affects receptor-binding affinity and specificity. The K119N mutation was also significantly detected in one specimen (3.1%) from a patient who had similar clinical symptoms as those of other patients during the second wave of the outbreak (Table 2). Neither mutation was detected in specimens of seasonal H1N1 in 2008, suggesting that further prospective study, as well as virological study, is required to understand the functions of these mutations.

HA residue 222 plays a critical role in the binding affinity of the galactose moiety of sialosaccharides for the RBS. The D222G mutation would result in a loss of interaction between galactose and K219 (Figure S4) because of the loss of charge and the side-chain. In turn, this would open up a cavity on the side of the RBS to accommodate the α2,3-linked receptor [11], [12]. Residue 223 is also important for the binding affinity of galactose and the terminal sialic acid of the receptor. The emergence of a positively charged and bulky Arginine(R) instead of Glutamine(Q) at this position would disturb the interaction between K219 and galactose, leading to decreased virus affinity for the α2,6-linked receptor (Figure S4) [13], [14]. Also, the homology modeling results showed that the double mutations, D187E and Q223R, would decrease virus affinity for the α2,6-linked receptor because the salt-bridge between E187 and R223 would lead to narrowing of the receptor binding pocket (data not shown).

In this study, we performed deep amplicon sequencing using Roche/454 GS-Junior technology. We compared the results ob<1?show=[dh][dh]?>tained by using deep sequencer with those obtained by the conventional Sanger method to evaluate this newly developed technology. Sequencing analysis using next-generation technology is necessary to reveal H1N1pdm genetic diversity in detail; our preliminary sequencing analysis using a conventional cloning approach failed to detect the D222G mutation in specimen #1 (Figure 1 and Table 2). The frequency of mutations such as D187E in specimens #1 and #2 and D222G in specimen #2 was not the same between GS-Junior technology and conventional Sanger sequencing (Figures 1, S1, S2, and S3, Tables 1 and 2). This discrepancy might be due to the different number of clones sequenced in each method. The proportion of HA sequences observed with D222G and Q223R in the #1 and #2 P6 and in the #3 P5 PCR products (Table 3) was consistent with results obtained by direct sequencing analysis using the ABI Sanger sequencer (Figure S6), suggesting that high-throughput amplicon sequencing analysis was highly quantitative.

The present study did not confirm whether viruses with the D222G and/or Q223R substitutions actually replicated in humans; however, the D222G and Q223R mutant viruses formed the dominant population when specimens were inoculated into embryonic chicken eggs (Table 3), indicating that the G222 and R223 mutants may be able to replicate in humans.

Several reports have shown that the D222G substitution was associated with severe, and sometimes fatal, cases of H1N1pdm [10], [15], [16], [17], [18]. It has also been reported that two different amino acids, D and E, may be polymorphic variants at position 222 [19], and that amino acids, G or N, would be present in potentially more pathogenic mutants or circulating variants [20]. However, the relevance of the mutation at position 222 to H1N1pdm pathogenesis remains unclear. In this study, D222E, D222N, and D222V variants were not detected, even when deep sequencing was performed (Table S1). D222G mutants were detected as a minor population even in mild cases; thus, every patient may have been exposed to D222G mutant viruses. Minority α2,3 sialic acid-tropic G222 mutants could be amplified in α2,3 sialic acid expressing cells in the lower respiratory tract of certain populations, which would result in severe to fatal respiratory illness in these populations. No conclusions can be drawn regarding the relationship between the D222G substitution and pathogenesis from the present study. Therefore, further study is required to determine whether G222 viruses were ever present as a major population in the lower respiratory tract of patients with severe or fatal pneumonia due to H1N1pdm infection.

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030946