Age-Related Pathology Associated with H1N1 A/California/07/2009 Influenza Virus Infection

Abstract

Influenza virus infection causes a spectrum of diseases, ranging from mild upper respiratory tract infection to severe lower respiratory tract infection, that can lead to diffuse alveolar damage, interstitial and airspace inflammation, or acute respiratory failure. Mechanisms instructing disease severity are not completely understood, but host, viral, and bacterial factors influence disease outcome. With age being one host factor associated with a higher risk of severe influenza, we investigated regional pulmonary distribution and severity of pneumonia after 2009 H1N1 influenza virus infection in newly weaned, adult, and aged ferrets to better understand age-dependent susceptibility and pathology. Aged ferrets exhibited greater weight loss and higher rates of mortality than adult ferrets, whereas most newly weaned ferrets did not lose weight but had a lack of weight gain. Newly weaned ferrets exhibited minimal pneumonia, whereas adult and aged ferrets had a spectrum of pneumonia severity. Influenza virus-induced pneumonia peaked earliest in adult ferrets, whereas aged ferrets had delayed presentation. Bronchial severity differed among groups, but bronchial pathology was comparable among all cohorts. Alveolar infection was strikingly different among groups. Newly weaned ferrets had little alveolar cell infection. Adult and aged ferrets had alveolar infection, but aged ferrets were unable to clear infection. These different age-related pneumonia and infection patterns suggest therapeutic strategies to treat influenza should be tailored contingent on age.

Figure 1

Weight loss and viral nasal wash titers after H1N1pdm09 virus infection. Naïve ferrets were infected with H1N1pdm09 virus (A/California/07/2009) and monitored for 2 weeks. A–C: Adult (A), aged (B), and newly weaned (C) ferrets were evaluated daily for weight loss. D: Mean weight loss for each group. E–G: Viral titers were determined from nasal washes collected at 1, 3, 5, and 8 days post infection. H: Mean viral titers for each group. Data are expressed as means ± SD virus titers.

Figure 2

Age-specific development of H1N1 influenza virus pneumonia. A–C: Pneumonia composite scores were assessed for 0, 1, 3, 5, 8, and 14 days post infection for each age group. D–S: Representative images from hematoxylin and eosin–stained sections are depicted for newly weaned (D, I, L, O, and R), adult (E, G, J, M, P, and S), and aged (F, H, K, N, and Q) ferrets during infection (0, 1, 3, 5, 8, and 14 days post infection). Data are expressed as mean scores (bars) and scores of individual animals (dots) (A–C). Original magnification, ×100 (D–S).

Figure 3

Comparison of global lung involvement and bronchial and alveolar pneumonia severity between each age group. A and D: The percentage of lung involvement was assessed in lung sections. Lung sections were scored as follows: 0, <10%; 1, 10% to 25%; 2, 26% to 50%; and 3, >50%. B, C, E, and F: Regional severity of pneumonia was assessed in the bronchi (B and E) and alveolar (C and F) spaces using the following scoring protocol: 0, normal; 1, mild pneumonia; 2, moderate pneumonia; and 3, severe pneumonia. Both the upper (A–C) and lower (D–F) left lung lobes were examined. *P < 0.05 adult versus aged; ^†P < 0.05 newly weaned versus aged; ^‡P < 0.05 newly weaned versus adult.

Figure 4

Age-specific H1N1 influenza virus lung infection. A–C: Infection composite scores were assessed for 0, 1, 3, 5, 8, and 14 days post infection for each age group. D–Q: Representative images of lung infection localized by influenza in situ hybridization are depicted for newly weaned (D, I, L, and O), adult (E, G, J, M, and P), and aged (F, H, K, N, and Q) ferrets during infection (0, 1, 3, 5, and 8 days post infection). Influenza matrix protein RNA transcripts appear as collections of black silver grains over cells counterstained with hematoxylin (blue). Data are expressed as mean scores (bars) and scores of individual animals (dots). Original magnification, ×100 (D–Q).

Figure 5

Comparison of regional lung infection severity between each age group. Graphs depict severity of influenza A virus infection in bronchial (A and C) and alveolar (B and D) spaces for the upper (A and B) and lower (C and D) lung lobes. The severity of influenza infection was determined by scoring of influenza virus in situ hybridization foci: 0, no definitive signal; 1, occasional focus; 2, focus in most fields; and 3, >1 focus per field. *P < 0.05 adult versus aged; ^†P < 0.05 newly weaned versus aged.

Figure 6

Comparison of submucosal gland (A–D) or trachea (E–H) infection severity between each age group. A and E: Submucosal gland (A) or trachea (E) infection severity. Severity of influenza infection was determined by scoring of influenza virus in situ hybridization (ISH) foci: 0, no definitive signal; 1, occasional focus; 2, focus in most fields; and 3, >1 focus per field. B–D: Representative images of submucosal gland infection of the lungs localized by influenza ISH are depicted for newly weaned (B) at 3 days post infection, adult (C) at 5 days post infection, and aged (D) ferrets at 8 days post infection. F–H: Representative images of trachea infection localized by influenza ISH are depicted for newly weaned (F), adult (G), and aged (H) ferrets at 5 days post infection. Influenza matrix protein RNA transcripts appear as collections of black silver grains over cells counterstained with hematoxylin (blue). Original magnification, ×100 (B–D and F–H). *P < 0.05 adult versus aged; ^†P < 0.05 newly weaned versus aged.

Supplemental Figure S1

Interlobar variability in pneumonia and influenza A virus lung infection. Low-power images of upper lung lobe (A, C, E, G, and I) and lower lung lobe (B, D, F, H, and J) infection localized by influenza in situ hybridization are depicted for adults (A–F) at 1, 3, and 5 days post infection (DPI) and aged ferrets (G–J) at 5 and 8 DPI. Influenza matrix protein RNA transcripts appear as collections of black silver grains over cells counterstained with hematoxylin (blue). Original magnification: ×10 (A, B, and E–J); ×50 (C and D).

Supplemental Figure S2

Comparison of global lung involvement and bronchial and alveolar pneumonia in male and female adult ferrets. A and B: The percentage of lung involvement was assessed in lung sections. Lung sections were scored as follows: 0, <10%; 1, 10% to 25%; 2, 26% to 50%; and 3, >50%. C–F: Regional severity of pneumonia was assessed in the bronchi (C and D) and alveolar (E and F) spaces using the following scoring protocol: 0, normal; 1, mild pneumonia; 2, moderate pneumonia; and 3, severe pneumonia. Both the upper (A, C, and E) and lower (B, D, and F) left lung lobes were examined.

Supplemental Figure S3

Comparison of regional lung infection severity between adult male and female ferrets. A–F: Graphs depict severity of influenza A virus infection in bronchial (A and B), alveolar (C and D), and submucosal glands (E and F) for the upper (A, C, and E) and lower (B, D, and F) lung lobes. G: Severity of influenza A virus infection in tracheal spaces. The severity of influenza infection was determined by scoring of influenza virus in situ hybridization (ISH) foci: 0, no definitive signal; 1, occasional focus; 2, focus in most fields; and 3, >1 focus per field.