Fig 1.
Pathogenicity of eq/J-K/08 H3N8 EIV in mice:
A:Percentage changes in body weight of EIV infected mice and negative control mice during the period of experiment (0–14 dpi). B:LDH level in pooled serum samples from EIV infected mice during the period of experiment. C:Humaral immune response following experimental EIV infection in BALB/c mice.
Fig 2.
Pathogenicity of eq/J-K/08 H3N8 EIV in mice:
A:Focal area of consolidation (red hepatization—arrow) of lung with moderate congestion at 5 dpi (n = 6). B: Degeneration of lining epithelial cells with diffuse goblet cell hyperplasia at 2 dpi X400. C: Degeneration of lining epithelial cells, severe degeneration of submucosal glandular epithelium and impacted lumen with neutrophils and necrotic tissue debris 3 dpi X400. D: Loss of cilia, with degeneration and necrosis of lining epithelium (arrow) of nasal mucosa at 2 dpi. E: Immunohistochemical staining (Fig 2C section) for EIV antigens in cytoplasm and nucleus of degenerated and necrotic lining epithelial cells of nasal turbinate (arrow) at 2 dpi (IIPT) X400. F: Severe degeneration of lining epithelial cells with goblet cell hyperplasia and presence of denuded ciliated epithelium along with lymphocytes in lumen (arrow) at 5 dpi X400. G: Goblet cell hyperplasia of lining epithelial cells with infiltrations of macrophages (arrow) and lymphocytes in lamina propria at 5 dpi X600 (n = 6).
Fig 3.
Pathogenicity of eq/J-K/08 H3N8 EIV in mice:Trachea (H&E):
A: Severe degeneration and necrosis of lining epithelial cells (arrow) at 2 dpi X400. B: Loss of cilia with necrosis of mucosal lining epithelium (arrow) at 2 dpi X1000. C: Tracheal lumen completely impacted with denuded epithelial cells with inflammatory exudates mixed with mucus at 2 dpi X100. D: Immunohistochemical staining for EIV positive antigens in cytoplasm of lining epithelial cells (arrow) at 3 dpi (IIPT) X600.E: Moderate degeneration of lining epithelial cells with severe infiltration of lymphocytes, macrophages and neutrophils in lamina propria at 5 dpi X400. F: Regeneration of lining epithelial cells with mild infiltrations of macrophages in lamina propria X400 at 7 dpi (n = 6).
Fig 4.
Pathogenicity of eq/J-K/08 H3N8 EIV in mice:Lung (H&E):
A: Early interstitial thickening with mild perivascular cuffing by inflammatory cells (arrow) at 1 dpi X100. B: Majority of bronchial epithelial cells shows hydropic degeneration and necrosis of lining epithelial cells (arrow) at 12hpi. C: Degenerated and necrosed bronchial epithelial cells (Fig 4C section) reveals EIV positive antigens in cytoplasm (arrow) at 12 hr post infection (IIPT) X400 (n = 6). D: Moderate degeneration and denudation of bronchial lining epithelial cells (arrow) with macrophage infiltration in interstitial spaces at 2 dpi X400. E: Immunohistochemical staining for EIV positive antigens (Fig 4D section) in degenerated and denuded bronchiolar epithelial cells (arrow) and interstitial macrophages at 2 dpi (IIPT) X400 (n = 6).
Fig 5.
Pathogenicity of eq/J—K/08 H3N8 EIV in mice: Lung (H&E):
A: Margination (arrow head), transmigration (arrow) and perivascular cuffing by neutrophils at 2 dpi X400. B: Necrotic bronchiolar epithelium with macrophages in bronchial lumen (arrow) at 5 dpi X400. C: Degeneration of lining epithelial cells (arrow) with mild peribronchiolar infiltration of lymphocytes at 5 dpi X400. Immunohistochemical localization of EIV antigens in mice lung. D: Distributions of EIV positive antigens in bronchiolar epithelial cells (arrow) at 5 dpi (Fig 5C section) (IIPT) X400 (n = 6). E: Severe consolidation of parenchyma with narrowing of bronchiolar lumen (arrow head) at 5 dpi X100 (n = 6). F: Resolution of lung lesion with mild thickening of alveolar septa and mild perivascular infiltrations at 14 dpi X100 (n = 6).
Fig 6.
Pathogenicity of eq/J-K/08 H3N8 EIV in mice:
Immunohistochemical localization of EIV antigens in mice lung. A: Intra-cytoplasmic EIV positive antigens in bronchiolar epithelial cells (arrow) at 3 dpi X1000. B: EIV positive interstitial macrophages (arrow) at 3 dpi X400. C: EIV positive antigens in cytoplasm of interstitial macrophages at 3 dpi (arrow) X1000 (n = 6). D: EIV positivity in bronchial lining epithelial cells (arrow) at 5 dpi (IIPT) X600 (n = 6).
Fig 7.
Pathogenicity of eq/J-K/08 H3N8 EIV in mice:
A: Intercellular space with influenza virions, budding and released from surface of the degenerating cells from trachea at 3 dpi. Irregularly shaped microvilli can be seen amongst the virions X10000. B: Ultra thin section of lung at 3 dpi, showing budding of influenza virions from degenerating cell along with disintegration of nuclear envelope and loss of organelle architecture X 10000. C: Budding and release of influenza virions from surface of a degenerating cell in lung X8000. D: Fragmentation of nucleus with degeneration of endoplasmic reticulum of epithelial cells of lung at 3 dpi X8000 (n = 6).
Table 1.
Comparison of EIV infection in BALB/c mice and natural host.
Fig 8.
A: Quantification and duration of EIV shedding through mice nostril (per ml of nasal washings) following experimental infection with EIV. B: Replication kinetics of EIV in mice lung (per gram of lung tissues) following experimental infection in BALB/c mice. C and D: Quantification of EIV genome copy numbers in nasal washings (140 μl) (n = 3) and lung tissues (25mg) (n = 6) following experimental EIV infection in BALB/c mice (n = 6).