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Yet, this average pattern is dominated by the presence of the H1N1/09 pandemic sequences.
A way to test this lability hypothesis is to estimate the TMRCA of the pandemic sequences.
Most of the pandemic sequences have an isoleucine at this position, just like the swine sequences isolated in 2009.
While pandemic sequences could have diverged as early as 2006 (Abdussamad and Aris-Brosou 2011), the first casualties were recorded back in March 2009 (Smith et al. 2009).
Unlike the TMRCA of the sampled H1N1 sequences, the emergence of the pandemic sequences shows a very consistent date across all segments and genes at 1.22 years before 2009, that is during the last semester of 2007 (SEM = 0.25 year).
In the rest of the text, we will denote the part of the tree that leads to the human 2009 pandemic sequences as the "pandemic clade", while all the other human sequences are part of the "non-pandemic clade".
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A more detailed analysis of the NA protein shows that the 2009 pandemic sequence is characterized by novel epitopes and by a particular substitution in loop 150, which is responsible for a nonadaptive structural change tightly associated with the emergence of the pandemic.
On the other hand, all the non-pandemic sequences and swine sequences isolated before 2009 have a valine, which is also the amino acid used by the 1918 H1N1 NA protein and that was found to distort the structure of the NA protein [ 27].
15 To determine amino acid changes that occurred as the influenza pandemic evolved, sequences were grouped semiannually, from 2009 to the first semester of 2014.
To assess the general impact of the pandemic H1N1/09 sequences on the results, the seasonality decompositions were conservatively limited to 1996-Q4/2008 (fourth quarter of 2008).
The sixth pandemic strain sequenced in this study was serotype O4 K68.
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