Genetic analysis of HA of influenza A (H1N1)pdm09 virus showed that this virus was a reassortant containing gene segments from ancestor viruses of human, swine and avian sources. Hence, Polymorphism at position 222 within the haemagglutinin (HA) molecule may have remarkable impact on viral host range, replication, and pathogenicity. It is worth noting that although the Asp222Gly mutation currently has not been associated with severe pandemic in humans.
An association between D222G and severity was initially proposed by Kilander et al. (2010)  and, since then, different studies in several countries  have found the D222G substitution to be more frequently associated with patients with severe pandemic influenza than in non-severe control cases. A recent study supported a role for this mutation in allowing the virus access to deeper lung tissue . Hese unusual viral attributes suggested that this new virus may possess some virulence characteristics similar to the highly pathogenic H5N1 or the 1918 pandemic influenza viruses.
This study used a conventional sequencing approach to analyze 50 H1N1pdm samples obtained from 2009 to 2011. Viruses with D222G substitution in HA protein have appeared sporadically and spontaneously in Tunisia since July 2009 . Although, 7% of them, found in severe cases in the present study, had the D222G substitution. This percentage is comparable to that found in Italy (4%) , in United Kingdom (6%) , in France (8%)  or in Spain (5%) , and lower than that published in Norway (18%) , and in Hong Kong (17.4%) . These differences with the Norwegian and Chinese study might be due to the reduced number of severe cases analyzed in that country. In Tunisia, this mutation was observed in circulating virus obtained from severe cases  and also from mild cases. The frequency of D222G substitution is higher in severe cases than mild cases. Although most of studies demonstrated the presence of D222G substitution in severe cases, it was also reported in mild cases [5, 22]. Therefore, the number of mild cases would need to be larger to determine whether mutant viruses are indeed circulating at a very low frequency also in non-severe cases.
The 222 G/E polymorphisms in the haemagglutinin (HA) gene of influenza A(H1N1)pdm09 virus have been associated with cases of mild to severe illness from different countries or geographical areas . Many retrospective analyses have found that cases bearing the D222G mutation were more likely to be associated with severe pneumonia, admission to intensive care facilities, and death . The majority of studies have reported that presence of D222G is sufficient to enhance virus replication and lethality in mouse models, with this effect ranging from modest to pronounced [25, 26]. Moreover, D222G substitution had been present in the Tunisian virus strain since pandemic season and throughout 2010–2011 season. Other groups have not observed substantial differences between wild-type and D222G viruses in mouse or ferret models , indicating the need for further investigation into the role of D222G in virulence of influenza A (H1N1)pdm09. The D222E was detected with less frequency than the D222G and only found in severe case patient. Despite the available virological, epidemiological and clinical information, the D222E substitution could confer more severity to the disease. The clinical significance of this mutation is still unclear . All above studies demonstrated that polymorphism of the HA protein, especially within the receptor binding domain, play a critical role in the binding preference and pathogenicity of influenza A(H1N1)pdm09 virus. Further study is warranted to elucidate the intriguing relationship between D222G substitution and severe disease.