Entry and release of transmissible gastroenteritis coronavirus are restricted to apical surfaces of polarized epithelial cells

Abstract

The transmissible gastroenteritis coronavirus (TGEV) infects the epithelial cells of the intestinal tract of pigs, resulting in a high mortality rate in piglets. This study shows the interaction of TGEV with a porcine epithelial cell line. To determine the site of viral entry, LLC-PK1 cells were grown on permeable filter supports and infected with TGEV from the apical or basolateral side. Initially after plating, the virus was found to enter the cells from both sides. During further development of cell polarity, however, the entry became restricted to the apical membrane. Viral entry could be blocked by a monoclonal antibody to the viral receptor aminopeptidase N. Confocal laser scanning microscopy showed that this receptor protein was present at both the apical and basolateral plasma membrane domains just after plating of the cells but that it became restricted to the apical plasma membrane during culture. To establish the site of viral release, the viral content of the apical and basolateral media of apically infected LLC-PK1 cells was measured by determining the amount of radioactively labelled viral proteins and infectious viral particles. We found that TGEV was preferentially released from the apical plasma membrane. This conclusion was confirmed by electron microscopy, which demonstrated that newly synthesized viral particles attached to the apical membrane. The results support the idea that the rapid lateral spread of TGEV infection over the intestinal epithelia occurs by the preferential release of virus from infected epithelial cells into the gut lumen followed by efficient infection of nearby cells through the apical domain.

Inhibition of porcine transmissible gastroenteritis virus (TGEV) replication in mini-pigs by shRNA

“…2 and 3) indicated that TGEV can replicate in several organs in pigs, but they mainly distribute in swine small intestine, for the amino peptidase N (APN), a TGEV major receptor in pigs, is abundantly expressed in the apical membrane of the small intestine epithelial cells, and which has a bigger quantity and higher activity in small intestine than other organs. Through the APN most of infectious TGEV invade host small intestine epithelial cells and initiate viral replication (Delmas et al, 1992;Rossen et al, 1994;Weingartl and Derbyshire, 1994). In our study, after the plasmids injected into mini-pigs via the precava, the reporter gene EGFP expressed abundantly in small intestine cells (data not shown).…”

“…It indicated that the plasmids were efficiently delivered into small intestine cells, the target of TGEV, by the method of precava injection. This may be a major reason why a small dose of shRNA-expressing plasmids (3 mg/piglet) could well protect mini-pigs from clinical symptoms and histopathology induced by TGEV infection (Delmas et al, 1992;Rossen et al, 1994;Weingartl and Derbyshire, 1995). The data also suggested that precava injection may be a good choice for treating animals with intestinal diseases as this route uses relatively short distance to reach small intestine from the precava.…”

Immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling

“…Porcine enteric coronaviruses replicate in IECs and are transmitted via a fecal-oral route. The viral entry and release for TGEV are restricted to the apical surface of polarized epithelial cells (Rossen et al, 1994). Experimental infection of newborn piglets with TGEV induces a strong and early IFN-␣ production in serum and intestinal secretions (La Bonnardiere and Laude, 1981).…”

Porcine aminopeptidase N mediated polarized infection by porcine epidemic diarrhea virus in target cells

“…2006; Cong and Ren, 2014). Epithelial cells grow with a polarized topology that involves the separation of the plasma membrane into apical and basolateral domains (Rossen et al, 1994;Cong and Ren, 2014). It has been demonstrated that the entry and release of several coronaviruses in polarized epithelial cells is restricted to the apical plasma membrane, e.g.…”