Identification of Pulmonary Infections With Porcine Rotavirus A in Pigs With Respiratory Disease

Abstract

While rotavirus (RV) is primarily known to cause gastroenteritis in many animals, several epidemiological studies have shown concurrent respiratory symptoms with fecal and nasal virus shedding. However, respiratory RV infections have rarely been investigated. By screening clinical samples submitted for diagnostic testing, porcine rotavirus A (RVA) was detected by quantitative reverse transcription PCR (qRT-PCR) in 28 out of 91 (30.8%) lungs obtained from conventionally reared pigs with respiratory signs. Among the positive cases, intensive RVA signals were mainly localized in alveolar macrophages (n = 3) and bronchiolar epithelial cells (n = 1) by RNAscope^® in situ hybridization (ISH). The signals of RVA in bronchiolar epithelial cells were verified by ISH with different probes, immunohistochemistry, and transmission electron microscopy. Furthermore, additional cases with RVA ISH-positive signals in alveolar macrophages (n = 9) and bronchial epithelial cells (n = 1) were identified by screening 120 archived formalin-fixed and paraffin-embedded lung samples using tissue microarrays. Overall, our study showed a high frequency of RVA detection in lungs from conventional pigs with respiratory disease. Further research is needed to determine if RVA infection in the respiratory epithelium correlates with nasal shedding of rotavirus and its contribution to respiratory disease.

Keywords: enteritis; extraintestinal rotavirus; interstitial pneumonia; porcine respiratory disease complex; rotavirus A.

Figure 1

Detection of rotavirus A (RVA) nucleic acid in the lung by in situ hybridization (ISH). Positive red signals were detected in round cells resembling macrophages in the interstitium (i) or alveolar macrophages and pneumonocytes lining the alveoli (a) (A) and (B). Focally, they were also detected in monocyte-macrophage lineage cells in peri-bronchial lymphoid aggregates (lym) (C) and bronchiolar epithelial cells (D). Images were taken under 400x magnification.

Figure 2

Verification of rotavirus A (RVA) infection in bronchiolar epithelial cells. Positive signal of RVA nucleic acid was detected by in situ hybridization (ISH) using probe targeting VP6 (A) and NSP3 (B). Positive signals of immunohistochemistry staining using antibody against RVA VP6 (C) and dsRNA (D), although weaker, were also detected in the serial sections obtained from the same case. No signal was detected by ISH using a probe targeting rotavirus B (E) (18). Serial section with routine hematoxylin and eosin stain (F) was performed. Images were taken under 400x magnification.

Figure 3

Visualization of rotavirus-like particles (VLP) in bronchial epithelial cells using transmission electron microscopy. VLP (arrows) ranging from 60 nm to 72 nm were observed near the apical border of the bronchiolar epithelial cells. Bar = 200 nm.