Development of molecular techniques for the detection and pathogenesis study of swine corona-and corona-like virus

Abstract

In situ hybridization (ISH) technique was first developed to detect and differentiate transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) in cell culture and tissue sections using ^®S-labled RNA probes. RNA probe generated from plasmid PSP.FP2 detected both TGEV and PRCV, whereas PSP.FP, probe detected only TGEV. The TGEV RNA was detected mainly within the enterocytes at the tips of villi and within a few crypt epithelial cells. The PRCV RNA was detected mainly In the bronchiolar epithelial cells and in lesser amount in type I and type II pneumocytes, alveolar macrophages and bronchial epithelial cells. Since the ISH technique using a radiolabeled probe is time consuming and not user friendly, the nonisotopic ISH technique using fluorescein-labeled RNA probe was developed. A rapid ISH technique using radiolabeled and fluorescein-labeled probes was able to decrease hybridization time from 20 hours to 2 hours without compromising the intensity of the signal and tissue morphology. By the rapid nonisotopic ISH technique, the entire procedure could be performed within about 7-8 hours. The mechanism of TGEV-induced cell death is not known. We demonstrated TGEV induced apoptosis in swine testes cell cultures by gel electrophoresis, electron microscopy, and terminal deoxytransferase digoxigenin-dUTP nick end labeling (TUNEL) technique. By electron microscopy, we showed that infected-ST cells from TGEV-inoculated wells were undergoing cell lysis, however, uninfected-ST cells were undergoing apoptosis. Double labeling technique also demonstrated that TGEV positive cells were negative for apoptosis and apoptotic cells were negative for TGEV RNA. Our results indicated that TGEV induced apoptosis in uninfected bystander cells, thus amplifying the cytopathic effect of TGEV. Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically emerging virus in swine. We demonstrated that PRRSV induced apoptosis both in vitro and in vivo and apoptotic cells were uninfected bystander cells. In the lungs of PRRSV-infected VIII pigs, the apoptotic cells were predominantly alveolar macrophages, lymphocytes, pulmonary intravascular macrophages, and type I and type II pneumocytes. In the lymph nodes of PRRSV-infected pigs, the apoptotic cells were predominantly lymphocytes and macrophages. A large number of macrophages and lymphocytes undergoing apoptosis might be the reason that PRRSV-infected pigs are susceptible to secondary infection.

Known Cellular and Receptor Interactions of Animal and Human Coronaviruses: A Review

“…TGEV is an enteropathogenic alphacoronavirus that causes diarrhoea in pigs [154]. Binding activity to APN is required for TGEV to initiate cellular infection [153] and the binding region of pAPN is believed to be AA residues 522-744 of the S glycoprotein [155][156][157]. Interestingly PRCV, an S gene deletion mutant of TGEV which displays altered in vivo tropism [90,158,159] binds pAPN comparably to TGEV [74].…”