D222G is the main substitution found in reports associated with severe or fatal cases of pdm09
[15, 19]. Here we found this substitution only in viruses isolated from severe cases; we also found a correlation between the risk of death and HA 222 variants. Low white blood cell (WBC) levels and low hemoglobin levels correlated with the presence of HA 222 variants, and suggested that the viruses harboring variants in HA 222 could be associated with a more severe respiratory disease. Not all severe or fatal cases had this mutation but its emergence could intensify the severity of the disease.
A recent study found a delayed clearance of virus in patients with severe disease
, which may represent an opportunity for the accumulation of different variants (including D222G) in patients. However in this study we found no correlation between duration of disease and emergence of D222 variants, supporting the argument that the substitutions appeared spontaneously and sporadically.
Respiratory tract cells expressing α2,3 receptors are more abundant deeper in the human airway, an observation that has led to the hypothesis that viruses with dual receptor specificity can replicate to higher titers in lungs, resulting in a more severe course of disease
. Recent studies suggest an increased receptor affinity of the 222G variant for ciliated bronchial epithelial cells, which may have an impact on disease severity
. Some studies indicate that such variants were also found more frequently in specimens obtained from the lower respiratory tract than from the nasopharynx
. Our findings indicate that the 222 variants including G222, N222, V222 or A222 were present in nasopharynx, showing their presence in the upper respiratory tract. Consistent with the presence of these variants in the upper respiratory tract, transmission of influenza virus with D222G from a patient with severe disease to a family member has been reported
. Other studies found that virus with D222G had acquired the ability to bind to SA α2,3Gal, retaining affinity for SA α2,6Gal, the preferential receptor of human viruses that is associated with attachment to cells of the upper respiratory tract
. Viruses with G222 do have dual tropism, perhaps resulting in more efficient infection of the lower respiratory tract, causing more severe disease. D222G, D222N, and D222E changes have been found in viruses isolated in different countries
[15, 19, 22, 24], but the D222V amino acid change has not been previously reported. A sequence of HA with A222 has been submitted to GenBank, but without any published clinical data. Changes in HA amino acid position 222 located in the receptor-binding cavity might potentially influence receptor binding of the influenza virus. Changing an acidic and polar amino acid (D) to neutral and non-polar (G, V and A) residue could affect the tropism of these new variants. Detection of HA 222 variants has been realized using both pyrosequencing and Sanger methods, resulting in different proportion of polymorphisms in that position
, in our case we found discrepancies also between both methodologies, due perhaps an enrichment of minority population during PCR used to amplify metagenomic sequencing library, especially given low amount of RNA used.
We also found that HA substitution G172R, which co-segregated with HA amino acid 222 variants, was present in fatal cases only. This substitution is located in the HA antigenic site Sa that could be implicated in antigenic changes of HA
. In fact, future amino acid substitutions in the antigenic sites of pdm09 HA (Gly172Glu) have been anticipated
. Furthermore, a HA variant with G172E was found in one fatal case
. The biological significance of the presence of both substitutions and their relationship with severity remains unknown.
The severity of the disease observed in human patients was generally consistent with the disease observed in mice. The virus INER047 isolated from a patient with a fatal outcome was lethal at 4 to 6 days PI in mice; and the virus INER111 that caused severe disease in a patient requiring mechanical ventilation was also lethal in mice at 4 and 5 days PI. However, the virus INER405 that had 222G in HA was of limited pathogenicity in mice. This virus was isolated from a patient who recovered after being hospitalized for one month showing an association between the virus and severe infection. However, the mouse study results suggest that additional determinants of pathogenicity in mammals likely exist, aside from 222G in HA, that might be responsible for severe disease in humans but are not determinants of disease in mice. This data suggests that there might be pathogenic factors affecting the clinical course of the disease in humans that have not been described. A recent report in an animal model showed that a pdm09 D222G virus caused more severe disease with fatal outcome in mice, compared with the wild type influenza virus
[23, 28], although experiments with ferrets did not support a causal link of D222G substitution with virulence
Here we report a high prevalence of pdm09 HA codon 222 variants in severe and fatal cases of pneumonia in Mexico City during the second wave of the 2009 influenza pandemic. Recent data confirmed an increase in the overall mortality in Mexico during the pandemic and also mortality due to respiratory and cardiovascular causes, especially in young and middle age individuals
. Our results support the suggestion that substitution at HA position 222 is associated with pdm09 pathogenicity, with variants appearing spontaneously during the two pandemic waves in Mexico. Some reports and sequence data have demonstrated that 222 variants appeared in the early phases of the pandemic, increased during 2009–2010 influenza season and have reduced to their present levels during 2010–2011 and 2011–2012 influenza season
[30, 31]. Bacterial infection or coexisting conditions do not appear to be major contributing factors to the severity (Table
2). We have identified additional viruses with 222 variants (D222V and D222A) in the same individual, but the biological importance of these substitutions in tropism and pathogenicity remains to be elucidated. The disease observed in animal models was generally concordant with the clinical outcome of patients, but additional determinants of virulence may contribute to the increased severity of disease observed in mammals and is a significant point for further investigation of viral determinants of severe disease in humans caused by pdm09 viruses.