MDCK cells were cultured in Eagle minimum essential medium as described (17). Influenza virus A/Netherlands/602/2009 was isolated from the first patient with pandemic (H1N1) 2009 virus infection in the Netherlands (18). Influenza virus A/Netherlands/213/2003 (seasonal influenza A [H3N2]) and influenza virus A/Netherlands/26/2007 (seasonal influenza A [H1N1]) were isolated from patients during epidemics in the Netherlands. After these viruses were passaged in MDCK cells 2×, all 8 gene segments were amplified by reverse transcription–PCR, cloned in a modified version of the bidirectional reverse genetics plasmid pHW2000 (19,20), and subsequently used to generate recombinant virus by reverse genetics as described elsewhere (19).

Mixtures of reassortant viruses were generated in 293T cells by using reverse genetics, by co-transfecting 8 plasmids that encode the pandemic (H1N1) 2009 virus genome together with 7 plasmids encoding the seasonal influenza A (H3N2) or seasonal influenza A (H1N1) virus genome. We omitted HA of the seasonal viruses to ensure that only reassortants containing the pandemic (H1N1) 2009 virus HA could arise, against which a large proportion of the human population is still immunologically naïve (21). The 293T-cell supernatants were passaged in quadruplicate under limiting dilution conditions by using 10-fold serial dilutions in MDCK cells 3× to enable selective outgrowth of viruses with high in vitro replication rates. After 3 passages, the genome composition of these viruses was determined by sequencing with conserved primers targeting noncoding regions of each gene segment. Reverse genetics was also used to produce specific reassortant viruses (pandemic [H1N1] 2009–seasonal influenza A [H1N1] basic polymerase [PB] 2, pandemic [H1N1] 2009–seasonal influenza A [H1N1] PB2 acidic polymerase [PA], pandemic [H1N1] 2009–seasonal influenza A [H3N2] NA, and pandemic [H1N1] 2009–seasonal influenza A [H3N2] NAPB1) by transfection of 293T cells and subsequent virus propagation in MDCK cells.

Multicycle replication curves were generated by injecting MDCK cells at a multiplicity of infection of 0.01 50% tissue culture infective dose (TCID₅₀) per cell in 2-fold (17). Virus titers from samples of inoculated MDCK cells, as well as nasal and throat swabs or homogenized tissue samples from inoculated ferrets, were determined by endpoint titration in MDCK cells, as described (22).

All animal studies were approved by an independent animal ethics committee. Experiments were performed under animal BioSafety Level 3+ conditions. The ferret model to test pathogenicity and transmission of pandemic (H1N1) 2009 virus was described previously (17,18). To study pathogenicity, 5 groups of 6 influenza virus–seronegative female ferrets (Mustella putorius furo) were inoculated intranasally with 10⁶ TCID₅₀ of wild-type pandemic (H1N1) 2009 virus, or the reassortant viruses pandemic (H1N1) 2009–seasonal influenza A (H1N1) PB2, pandemic (H1N1) 2009–seasonal influenza A (H1N1) PB2PA, pandemic (H1N1) 2009–seasonal influenza A (H3N2) NA, and pandemic (H1N1) 2009–seasonal influenza A (H3N2) NAPB1, divided between both nostrils (2 × 250 μL). In the transmission experiment, 4 female ferrets for wild-type pandemic (H1N1) 2009 virus and 2 ferrets for each reassortant virus were individually housed in transmission cages and inoculated intranasally with 10⁶ TCID₅₀ of virus divided between both nostrils (2 × 250 μL). Animal daily weights were used as an indicator of disease.

Immunohistochemical testing and pathologic examination were performed by using lungs of inoculated ferrets. For each virus, 3 ferrets were euthanized at 3 and 7 days postinoculation (dpi) by exsanguination. Necropsies and tissue sampling were performed according to standard protocol. After fixation in 10% neutral-buffered formalin and embedding in paraffin, samples were sectioned at 4 μm and stained with an immunohistochemical method by using a mouse monoclonal antibody against the nucleoprotein of influenza virus A (23). Influenza virus antigen expression in lung sections was scored for bronchial surface epithelium, bronchial submucosal gland epithelium, bronchiolar epithelium, alveolar type I pneumocytes, and alveolar type II pneumocytes. Scoring was categorized as 0, no positive cells; 1, few positive cells; 2, moderate number of positive cells; and 3, many positive cells. Serial lung sections were stained with hematoxylin and eosin for detection and description of pathologic changes. Samples were scored for influenza virus–associated inflammation in bronchi (bronchitis), bronchial submucosal glands (bronchoadenitis), bronchioles (bronchiolitis), and alveoli (alveolitis). Scoring of severity of inflammation was 0, no inflammation; 1, mild inflammation; 2, moderate inflammation; and 3, marked inflammation. Researchers who examined the sections had no knowledge of the identity of the ferrets.

The proportion of gene segments 1–8 (except HA) analyzed was ≈60%, 60%, 65%, 90%, 95%, 100%, and 100%, respectively. Minor virus variants were not detected. No point mutations were observed in the proportions of the genome analyzed. Upon pandemic (H1N1) 2009–seasonal influenza A (H1N1) transfection and passaging, 3 reassortants contained the PB2 gene of seasonal influenza virus A (H1N1), 2 of which had also incorporated the seasonal (H1N1) PA gene. All 4 pandemic (H1N1) 2009–seasonal influenza A (H3N2) virus reassortants had the NA gene of seasonal influenza A (H3N2), and 3 of 4 reassortants also incorporated the seasonal influenza A (H3N2) PB1 gene (Table 1).

The replication kinetics of pandemic (H1N1) 2009–seasonal influenza A (H1N1) PB2 and pandemic (H1N1) 2009–seasonal influenza A (H1N1) PB2PA were similar to those of wild-type pandemic (H1N1) 2009 virus, and the pandemic (H1N1) 2009–seasonal influenza A (H3N2) NA and pandemic (H1N1) 2009–seasonal influenza A (H3N2) NAPB1 reassortant viruses displayed slightly higher virus titers at 24 and/or 48 h after inoculation, with a maximum difference in virus titer of 1.0 log₁₀ TCID₅₀ (Figure 1). The fact that each reassortant virus replicated at least at the same rate as the wild-type pandemic (H1N1) 2009 virus agrees with results from the in vitro selection experiment described above.

The mean maximum weight loss was 7% for animals inoculated with the pandemic (H1N1) 2009 virus. Animals inoculated with pandemic (H1N1) 2009–seasonal influenza A (H1N1) PB2PA, pandemic (H1N1) 2009–seasonal influenza A (H1N1) PB2, pandemic (H1N1) 2009–seasonal influenza A (H3N2) NAPB1 and pandemic (H1N1) 2009–seasonal influenza A (H3N2) NA had a maximum weight loss of 4%, 2%, 2%, and 6%, respectively (data not shown).

Nose and throat swabs were collected daily, and virus titers were determined. Infectious virus shedding continued until 6–7 days dpi from noses (Figure 2, panels A and C) and throats (Figure 2, panels B and D) of most inoculated animals. Total virus shedding from the nose, as calculated from the area under the curve for ferrets in the experiment for 7 days (n = 3), was significantly lower in animals inoculated with the pandemic (H1N1) 2009–seasonal influenza A (H1N1) PB2 reassortant virus (p = 0.003 by t test) and significantly higher in the animals inoculated with the pandemic (H1N1) 2009–seasonal influenza virus A (H3N2) NA (p = 0.023 by t test), than in animals inoculated with wild-type pandemic (H1N1) 2009 virus. Total virus shedding from the throat, as calculated from the area under the curve for ferrets in the experiment for 7 days, was not significantly different between the groups of ferrets.

At 3 and 7 dpi, 3 ferrets from each group were euthanized, and samples from nasal turbinates, trachea, and lungs were collected for virologic examination. At 7 dpi, virus was undetectable or detected at only very low levels in these samples from all groups of ferrets. At 3 dpi, no virus or relatively low virus titers were detected in the lungs and trachea respectively, of ferrets inoculated with pandemic (H1N1) 2009–seasonal influenza A (H1N1) reassortant viruses (Figure 3, panels A and B), and titers in the nasal turbinates were similar to those for wild-type pandemic (H1N1) 2009 virus (Figure 3, panel C). These data indicate that both pandemic (H1N1) 2009–seasonal influenza A (H1N1) viruses were attenuated with respect to replication in the lower respiratory tract of ferrets.

At 3 dpi, virus was detected in the lungs, trachea, and nasal turbinates of ferrets inoculated with pandemic (H1N1) 2009–seasonal influenza A (H3N2) NAPB1 and pandemic (H1N1) 2009–seasonal influenza A (H3N2) NA viruses at approximately the same levels as upon inoculation with wild-type pandemic (H1N1) 2009 virus (Figure 3, panels D–F). Virus titers detected in the lungs and trachea of animals inoculated with pandemic (H1N1)–seasonal influenza virus A (H3N2) NA at 3 dpi were 1.0 log₁₀ TCID₅₀ higher than those in animals inoculated with wild-type pandemic (H1N1) 2009 virus (not statistically significant). These data indicate that both pandemic (H1N1) 2009–seasonal influenza A (H3N2) viruses tested were not attenuated in ferrets. If anything, shedding of pandemic (H1N1)–seasonal influenza A (H3N2) NA virus from the nose (Figure 2, panel C), lungs (Figure 3, panel D), and trachea (Figure 3, panel E) was higher than shedding of wild-type pandemic (H1N1) 2009 virus.

At 7 dpi, virus antigen expression was undetectable in lung tissue of any of the euthanized ferrets, and lesions were absent or resolving. At 3 dpi, neither viral antigen expression nor lesions were detected in lungs of ferrets inoculated with pandemic (H1N1) 2009–seasonal influenza A (H1N1) PB2. Only 1 of 3 ferrets inoculated with pandemic (H1N1) 2009 had scant virus antigen expression and mild associated lesions in bronchial submucosal glands and bronchioles at 3 dpi (Table 2; Figure 4). In contrast, all 3 ferrets inoculated with pandemic (H1N1) 2009–seasonal influenza A (H3N2) NA had moderate to abundant virus antigen expression in bronchial submucosal glands, bronchioles, or both, associated with moderate to marked inflammation (Table 2; Figure 4). Virus antigen expression and associated lesions in the lungs of ferrets inoculated with pandemic (H1N1) 2009–seasonal influenza A (H3N2) PB1NA were intermediate between those of wild-type pandemic (H1N1) 2009 and pandemic (H1N1)–seasonal influenza A (H3N2) NA (Table 2).

Cell types in which virus antigen expression was detected were ciliated epithelial cells of bronchi, epithelial cells of bronchial submucosal glands, ciliated and nonciliated cells of bronchioles, and both squamous and cuboidal epithelial cells (interpreted as type I and type II pneumocytes, respectively) of alveoli (Figure 4). Virus antigen expression was also seen in desquamated epithelial cells and cell debris in lumina of above tissues.

Lesions associated with virus antigen expression can be categorized as acute, focal or multifocal, necrotizing bronchitis, bronchoadenitis, bronchiolitis, and alveolitis. These lesions were characterized by degeneration and necrosis of epithelial cells, infiltration of the affected tissues and their lumina by many neutrophils and few eosinophils, and exudation of edema fluid and fibrin into tissue lumina.

Transmission of pandemic (H1N1) 2009 and reassortant influenza viruses through aerosol or respiratory droplets was tested in the ferret model. Ferrets in groups of 4 for pandemic (H1N1) 2009 virus and 2 for the reassortant viruses were inoculated intranasally with 10⁶ TCID₅₀ of virus. At 1 dpi, an uninfected ferret was placed in a cage adjacent to each inoculated ferret. All viruses were transmitted from the inoculated to the uninfected ferrets in 4/4 ferrets for pandemic (H1N1) 2009 virus and 2/2 ferrets for each of the reassortant viruses. The first day of virus detection in the previously uninfected animals was 2 days post exposure, similar for all viruses tested.