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A (H1N1) pdm09 HA D222 variants associated with severity and mortality in patients during a second wave in Mexico

Joel A Vazquez-Perez1, Pavel Isa2, Darwyn Kobasa45, Christopher E Ormsby1, Jose E Ramírez-Gonzalez3, Damaris P Romero-Rodríguez1, Charlene Ranadheera45, Yan Li45, Nathalie Bastien4, Carissa Embury-Hyatt6, Elizabeth González-Duran3, Gisela Barrera-Badillo3, Yuria Ablanedo-Terrazas1, Edgar E Sevilla-Reyes1, Marina Escalera-Zamudio2, Ana G Cobián-Güemes2, Irma Lopez3, Joanna Ortiz-Alcántara3, Celia Alpuche-Aranda3, Jose R Perez-Padilla1 and Gustavo Reyes-Terán17*

Author Affiliations

1 Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico

2 Instituto de Biotecnología, UNAM, Cuernavaca, Morelos, Mexico

3 Instituto Nacional de Diagnóstico y Referencia Epidemiológica, Mexico City, Mexico

4 National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada

5 Department of Medical Microbiology, University of Manitoba, Manitoba, Canada

6 National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Ottawa, Canada

7 Centro de Investigaciones en Enfermedades Infecciosas, Instituto Nacional de Enfermedades Respiratorias, Tlapan 4502, CP 14080, Mexico City, Mexico

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Virology Journal 2013, 10:41  doi:10.1186/1743-422X-10-41

Published: 31 January 2013



Pandemic type A (H1N1) influenza arose in early 2009, probably in Mexico and the United States, and reappeared in North America in September for seven more months. An amino acid substitution in the hemagglutinin (HA), D222G, has been reported in a significant proportion of patients with a severe and fatal outcome. We studied the prevalence of HA222 substitutions in patients in Mexico during the second wave and its association with clinical outcome and pathogenicity in a mouse model.


The nucleotide sequences of hemagglutinin (HA) from viruses collected from 77 patients were determined including 50 severe and fatal cases and 27 ambulatory cases. Deep sequencing was done on 5 samples from severe or fatal cases in order to determine the quasispecies proportion. Weight loss and mortality due to infection with cultured influenza viruses were analyzed in a mouse model.


Viruses from 14 out of 50 hospitalized patients (28%) had a non aspartic acid residue at the HA 222 position (nD222), while all 27 ambulatory patients had D222 (p = 0.0014). G222 was detected as sole species or in coexistence with N222 and D222 in 12 patients with severe disease including 8 who died. N222 in coexistence with D222 was detected in 1 patient who died and co-occurrence of A222 and V222, together with D222, was detected in another patient who died. The patients with a nD222 residue had higher mortality (71.4%), compared to the group with only D222 (22.2%, p = 0.0008). Four of the 14 viruses from hospitalized patients were cultured and intranasally infected into mice. Two viruses with G222 were lethal while a third virus, with G222, caused only mild illness in mice similar to the fourth virus that contained D222.


We confirm the elevated incidence of HA222 (H1N1)pdm09 variants in severe disease and mortality. Both clinical and mouse infection data support the idea that nD222 mutations contribute to increased severity of disease but additional determinants in disease outcome may be present.