Prevalence of enteric coronavirus infection among Thais and characterization of their monoclonal antibodies

Abstract

An enzyme-linked immunosorbent assay (ELISA) for detection of coronaviruses in fecal specimens was compared to a protein A-coated grid immunosorbent electron microscopy technique (PA-CGT). All fecal samples were primarily screened by the ELISA and confirmed by an ELISA blocking test. The randomly selected ELISA blocking test negative and positive samples were then determined for the presence of virion by PA-CGT. Of 39 ELISA blocking test negative specimens, 38 (97.4%) were still negative by PA-CGT whereas the viruses were seen in only 26 out of 32 (81.2%) ELISA blocking test positive specimens. ELISA blocking test was superior to PA-CGT for detecting human enteric coronaviruses. The prevalence of enteric coronavirus infection, among the Thai populations in Bangkok and Khon Kaen province, was then studied using the ELISA blocking test. The results indicated that coronaviruses were present in fecal samples from both children and adults with or without diarrhea. It is interesting to note that, in Bangkok, the viruses were more frequently observed in the apparently healthy individuals (8.59%) than in the patients with diarrhea (2.56%). Among non-diarrheal populations, coronaviruses were frequently observed in stools from children over one year of age and adults but not in children aged less than one year whereas in patients with diarrhea coronavirus infection started in the early neonatal life. The rate of coronavirus excretion in both groups did not increase with age and some apparently healthy subjects excreted the viruses in the feces for at least seven months. The results also suggested that chronic parasitic infection but not rotavirus and/or bacterial infections of the enteric tract might influence excretion of enteric coronaviruses in the feces. Moreover, the presence of coronaviruses in the normal population may be responsible for a long term silent infection of the enteric tract and chronic infection possibly ensued. Monoclonal antibodies against human enteric coronaviruses (HECV-Ric13) and bovine enteric coronavirus (BCV-F16) were obtained and characterized by SDS-polyacrylamide electrophoresis (SDS-PAGE) of the immunoprecipitates derived from reaction between these antibodies and (125)I-labeled purified HECV-Ric13 and BCV-F16 which contained five polypeptides of molecular weights 98K, 63K, 55K, 47K and 22K. Twelve clones of the hybridomas secreting antibodies against HECV-Ric 13 were obtained. Among these, 10 monoclonal antibodies reacted to the 55K capsid protein while the other two reacted to the high molecular weight peplomeric protein. These antibodies also reacted with the proteins of BCV-F16. Only one monoclonal antibody directed against BCV-F16 was obtained; it reacted to the 55K protein of BCV-F16 and HECV-Ric13.