Adenovirus 14p1 Immunopathogenesis during Lung Infection in the Syrian Hamster

Abstract

Adenovirus (Ad) infections are usually mild and self-limited, with minimal inflammatory responses. During worldwide outbreaks, Ad14p1, an emerging Ad14 variant, has caused severe pulmonary disease, including acute respiratory distress syndrome (ARDS). This increased pathogenicity of Ad14p1 is not completely understood. In initial studies, we observed that infection of Syrian hamsters with Ad14p1 can cause a patchy bronchopneumonia, with an increased intensity of inflammation, compared to wild type Ad14 infection. The current study compared the dynamics of the immunopathogenesis of Ad14 and Ad14p1 infection of hamster lungs through the first two weeks after infection. Little difference was seen in infection-induced inflammation at day 1. Beginning at day 3, Ad14p1-infected hamsters showed marked inflammation that continued through to day 7. The inflammation began to resolve by day 10 but was still detectable at day 14. In contrast, Ad14-infected hamsters showed little inflammation during the 14-day period of observation. Inflammatory cell type analysis revealed that, at day 1, hamsters infected with either virus had predominantly neutrophil infiltration that began to resolve by day 3. However, at day 5, Ad14p1-infected hamsters had a second wave of neutrophil infiltration that was accompanied by edema which persisted to a variable extent through to day 10. These differences were not explained by an increased Ad14p1 replication rate, compared with Ad14 in vitro, but there was prolonged persistence of Ad14p1 in hamster lungs. There were differences in lung tissue cytokine and chemokine responses to Ad14p1 vs. Ad14 infection that might account for the increased leukocyte infiltrates in Ad14p1-infected hamsters. This animal model characterization provides the basis for future translational studies of the viral genetic mechanisms that control the increased immunopathogenesis of the emergent, Ad14p1 strain.

Keywords: Syrian hamster; acute lung injury; acute respiratory distress syndrome; adenovirus.

Figure 1

Effects of Ad14 and Ad14p1 on Syrian hamster lung pathology. H&E staining of Syrian hamster lungs post infection with 5 × 10⁹ genomes/animal via intratracheal instillation. Slides were scanned with Pathscan Enabler IV at 3600 dpi (Meyer Instruments, Houston, TX, USA) and are representative of four hamsters. Bar = 1 mm.

Figure 2

Intensity of Ad14 and Ad14p1 induced lung inflammation. H&E sections from Ad14- and Ad14p1-infected hamsters were scored by a pathologist for inflammation based on the lung allograft rejection scoring standards for (A) A-component and (B) B-component. H&E sections from Ad14- and Ad14p1-infected hamsters were scored from the same slides by a pathologist according the guidelines described in the methods section to determine regional pneumonitis: (C) Intra-alveolar (D) alveolar, (E) bronchial, (F) interstitial, and (G) peribronchial inflammatory scores. H&E sections from an Ad14p1-infected hamster at day 5 post infection. Representative 40× images of (A) Grade A3, A-component score, (B) high-grade B-component score, and (C) G3 intra-alveolar score. n = 4, Mean ± SD.

Figure 3

Effects of Ad14 and Ad14p1 on hamster lung cells. Airway epithelial cell changes were assessed by evaluating (A) bronchiolar epithelial cell damage, (B) bronchiolar epithelial cell hyperplasia, and (C) Type II pneumocyte hyperplasia. Scoring criteria are explained in the methods section. n = 4, Mean ± SD.

Figure 4

Inflammatory cell composition of Ad14- and Ad14p1-induced pneumonias. Differential cell counts for macrophages (A), neutrophils (B), and lymphocytes (C) were determined at the periphery of pneumonias by a blinded pathologist and expressed as the percentage of total inflammatory cells (mean ± SEM, n = 4). Immunohistochemistry was used to confirm the presence of macrophages with anti-CD63, neutrophils with anti-MPO, and lymphocytes with anti-CD3ε. Images (100×) are representative of four hamsters.

Figure 5

Effects of Ad14 and Ad14p1 on inflammatory cell composition of bronchoalveolar lavage (BAL) fluid. Syrian hamsters were infected with 5 × 10⁹ genomes/animal via intratracheal instillation. BAL was performed on the days indicated, and cytospins were performed to determine differential cell counts. Macrophages (A), neutrophils (B), lymphocytes (C), and monocytes (D) are expressed as the percentage of total inflammatory cells (mean ± SEM). A one-way ANOVA was performed with p-values determined by a post-hoc Sidak test (n = 4, *** p < 0.001, ** p < 0.01, * p < 0.05).

Figure 6

Permissive Ad14 and Ad14p1 infection in Syrian hamster lungs. (A) Viral loads were assessed at days 1, 3, 5, 7, 10, and 14 post infection by qPCR from homogenates of one of the right lung lobes and expressed as genomes/g tissue. Mean ± SEM, n = 4, one-way ANOVA followed by Holm–Sidak, * p < 0.05. (B) Presence of smudge cells (arrowhead) and inclusion bodes (arrow) in an H&E stained section of the lung of an Ad14p1-infected hamster at day 7 post infection (100×). (C) Immunohistochemistry staining of Ad14p1 with anti-hexon antibody (adenovirus 20/11 and 2/6; Cell Marque) in infected hamster lungs at day 5 and 10 post infection. Images are representative of four hamsters per time point.

Figure 7

Cytokine expression in the lungs during Ad14 and Ad14p1 infection. (A) IL-1β, (B) TNF-α, (C) MIP-1α, and (D) IP-10 gene expression were determined by RT-qPCR of RNA from one of the right lung lobes of infected hamsters at the indicated times post infection. Fold change was determined by normalizing to RPL-18 expression and then by the 2 ΔΔCt method against uninfected control animals. Mean ± SEM, n = 4, one-way ANOVA followed by Holm–Sidak (*** p < 0.001, ** p < 0.01, * p < 0.05).