Possible increased pathogenicity of pandemic (H1N1) 2009 influenza virus upon reassortment

Abstract

Since emergence of the pandemic (H1N1) 2009 virus in April 2009, three influenza A viruses-seasonal (H3N2), seasonal (H1N1), and pandemic (H1N1) 2009-have circulated in humans. Genetic reassortment between these viruses could result in enhanced pathogenicity. We compared 4 reassortant viruses with favorable in vitro replication properties with the wild-type pandemic (H1N1) 2009 virus with respect to replication kinetics in vitro and pathogenicity and transmission in ferrets. Pandemic (H1N1) 2009 viruses containing basic polymerase 2 alone or in combination with acidic polymerase of seasonal (H1N1) virus were attenuated in ferrets. In contrast, pandemic (H1N1) 2009 with neuraminidase of seasonal (H3N2) virus resulted in increased virus replication and more severe pulmonary lesions. The data show that pandemic (H1N1) 2009 virus has the potential to reassort with seasonal influenza viruses, which may result in increased pathogenicity while it maintains the capacity of transmission through aerosols or respiratory droplets.

Figure 1

Replication of wild-type and reassortant pandemic (H1N1) 2009 viruses in MDCK cells. MDCK cells were injected in duplicate with 0.01 50% tissue culture infective dose (TCID₅₀) per cell of each virus: black, wild-type pandemic (H1N1) 2009; red, reassortant pandemic (H1N1) 2009–seasonal influenza (H1N1) basic polymerase (PB) 2 acidic polymerase; blue, reassortant pandemic (H1N1) 2009–seasonal influenza (H1N1) PB2; green, reassortant pandemic (H1N1) 2009–seasonal influenza (H3N2) PB1 neuraminidase (NA); orange, reassortant pandemic (H1N1)–seasonal influenza (H3N2) NA. Supernatant samples were harvested 6, 12, 24 and 48 h after injection. Supernatant samples were titrated in MDCK cells. Geometric mean titers and standard deviation were calculated from 2 independent experiments.

Figure 2

Virus shedding from the nose and throat of ferrets inoculated with wild-type and reassortant pandemic (H1N1) 2009 viruses. Virus shedding from nose (A, C) and throat (B, D) is shown for pandemic (H1N1) 2009–seasonal influenza (H1N1) (A, B) and pandemic (H1N1) 2009–seasonal influenza (H3N2) (C, D) reassortant viruses. Black, wild-type pandemic (H1N1) 2009; red, pandemic (H1N1) 2009–seasonal influenza (H1N1) basic polymerase (PB) 2 acidic polymerase; blue, pandemic (H1N1) 2009–seasonal influenza (H1N1) PB2; green, pandemic (H1N1) 2009–seasonal influenza (H3N2) PB1 neuraminidase (NA); orange, pandemic (H1N1)–seasonal influenza (H3N2) NA. Geometric mean titers are shown; error bars indicate SD. The lower limit of detection is 0.5 log₁₀ 50% tissue culture infective dose/mL (TCID₅₀/mL). After day 3, only 3 animals remained in each group.

Figure 3

Virus detection in respiratory tissues of ferrets inoculated with wild-type and reassortant pandemic (H1N1) 2009 viruses. Virus detection in lungs (A, D), trachea (B, E), and nasal turbinates (C, F) is shown for pandemic (H1N1) 2009–seasonal influenza (H1N1) (A–C) and pandemic (H1N1) 2009–seasonal influenza (H3N2) (D–F) reassortant viruses. Black, wild-type pandemic (H1N1) 2009; red, pandemic (H1N1) 2009–seasonal influenza (H1N1) basic polymerase (PB) 2 acidic polymerase; blue, pandemic (H1N1) 2009–seasonal influenza (H1N1) PB2; green, pandemic (H1N1) 2009–seasonal influenza (H3N2) PB1 neuraminidase (NA); orange, pandemic (H1N1)–seasonal influenza (H3N2) NA. Three ferrets of each group were euthanized at 3 and 7 days postinoculation. Geometric mean titers are shown; error bars indicate SD. The lower limit of detection is 0.5 log₁₀ 50% tissue culture infective dose/mL (TCID₅₀/mL).

Figure 4

Examples of virus antigen expression and severity of lesions in different tissues of the lungs of ferrets. A) Bronchial surface; B) bronchial submucosal gland; C) bronchiole; D) alveolus. Two of 3 ferrets inoculated with wild-type pandemic (H1N1) 2009 virus had neither virus antigen expression (first column) nor associated lesions (second column) in the lung at day 3 postinoculation. In contrast, all 3 ferrets inoculated with reassortant pandemic (H1N1) 2009–seasonal influenza (H3N2) virus neuraminidase had virus antigen expression in bronchi, bronchial submucosal glands, bronchioles, and alveoli (third column), associated with epithelial degeneration and necrosis and infiltration of inflammatory cells, predominantly neutrophils (fourth column). IHC, immunohistochemistry; H&E, hematoxylin and eosin stain. Original magnification ×400.