Porcine Hemagglutinating Encephalomyelitis Virus Infection In Vivo and Ex Vivo

Abstract

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a Betacoronavirus that causes vomiting and wasting disease and/or encephalomyelitis in suckling pigs. This study characterized PHEV infection, pathogenesis, and immune response in caesarean-derived, colostrum-deprived (CDCD) neonatal pigs. Infected animals developed mild respiratory, enteric, and neurological clinical signs between 2 to 13 days post oronasal inoculation (dpi). PHEV did not produce viremia, but virus shedding was detected in nasal secretions (1-10 dpi) and feces (2-7 dpi) by RT-qPCR. Viral RNA was detected in all tissues except liver, but the detection rate and RT-qPCR Ct values decreased over time. The highest concentration of virus was detected in inoculated piglets necropsied at 5 dpi in turbinate and trachea, followed by tonsils, lungs, tracheobronchial lymph nodes, and stomach. The most representative microscopic lesions were gastritis lymphoplasmacytic, moderate, multifocal, with perivasculitis, and neuritis with ganglia degeneration. A moderate inflammatory response, characterized by increased levels of IFN-α in plasma (5 dpi) and infiltration of T lymphocytes and macrophages was also observed. Increased plasma levels of IL-8 were detected at 10 and 15 dpi, coinciding with the progressive resolution of the infection. Moreover, a robust antibody response was detected by 10 dpi. An ex vivo air-liquid CDCD-derived porcine respiratory cells culture (ALI-PRECs) system showed virus replication in ALI-PRECs and cytopathic changes and disruption of ciliated columnar epithelia, thereby confirming the tracheal epithelia as a primary site of infection for PHEV. IMPORTANCE Among the ∼46 virus species in the Family Coronaviridae (2019), many of which are important pathogens of humans and 6 of which are commonly found in pigs, porcine hemagglutinating encephalomyelitis remains one of the least researched. The present study provided a comprehensive characterization of the PHEV infection process and immune responses using CDCD neonatal pigs. Moreover, we used an ex vivo porcine respiratory cells culture (ALI-PRECs) system resembling the epithelial lining of the tracheobronchial region of the porcine respiratory tract to demonstrate that the upper respiratory tract is a primary site of PHEV infection. This study provides a platform for further multidisciplinary studies of coronavirus infections.

The Betacoronavirus PHEV Replicates and Disrupts the Respiratory Epithelia and Upregulates Key Pattern Recognition Receptor Genes and Downstream Mediators, Including IL-8 and IFN-λ

“…In a previous study ( 6 ), we reported the detection of significant PHEV RNA levels in tracheal tissues collected from pigs experimentally inoculated with PHEV and necropsied on day postinfection (dpi) 5. As a continuation, in the present study, we performed transcriptome analysis (45 target genes) of these tracheal tissues in comparison to their corresponding mock-inoculated pigs as the negative control.…”

“…Actually, the virus was first isolated in 1970 from the nasal cavity of apparently healthy pigs during a routine diagnostic survey ( 8 ), and viral antigens were detected inside the cytoplasm of the epithelial cells lining nasal mucosa, bronchi, and bronchioles ( 12 ). Our group further demonstrated that, under in vivo and ex vivo experimental conditions, PHEV (67N strain) replicates in the upper respiratory tract epithelium and tonsil of cesarean-derived, colostrum-deprived (CDCD) neonatal pigs ( 6 ). Therefore, as for many other pathogens with respiratory tropism, respiratory epithelial cells could play a vital role in initiating and shaping the crucial defensive immunological processes during PHEV infections.…”

“…The epithelial surfaces of ALI-PRECs also expressed different host viral receptors such as sialic acids ( 37 ), ACE2 ( 38 ), CEACAM1 ( 39 ), DPP4 ( 40 ), and APN ( 41 ), widely used by influenza viruses and coronaviruses. Consequently, ALI cell cultures support viral infections, and they have been used to study host-virus interactions ( 6 , 19 – 23 ).…”

“…PHEV is the causative agent of vomiting and wasting disease (VWD) and/or encephalomyelitis in suckling pigs ( 3 – 5 ). Recent in vivo infection studies from our group further demonstrated that PHEV-affected regions were characterized by a variable degree of lymphocyte and macrophage infiltration ( 6 ). However, the mechanisms causing this inflammatory infiltration are not completely understood.…”

“…Different experimental studies ( 6 – 10 ) and field outbreak investigations ( 4 , 10 , 11 ) described PHEV-associated respiratory signs early after infection. More recently, PHEV has been associated with respiratory disease in pigs ( 11 ).…”