Comparison of immunofluorescence with monoclonal antibodies and RT-PCR for the detection of human coronaviruses 229E and OC43 in cell culture

Abstract

Human coronaviruses, with two known serogroups named 229E and OC43, cause up to one third of common colds and may be associated with serious diseases such as nosocomial respiratory infections, enterocolitis, pericarditis and neurological disorders. Reliable methods of detection in clinical samples are needed for a better understanding of their role in pathology. As a first step in the design of such diagnostic procedures, the sensitivities and specificities of two viral diagnostic assays were compared in an experimental cell culture model: an indirect immuno-fluorescence assay using monoclonal antibodies and reverse transcriptase-polymerase chain reaction amplification of viral RNA from infected cells. Immunofluorescence detected human coronaviruses in cells infected at a MOI as low as 10(-2) (log TCID50/ml = 4.25 for HCV-229E and 2.0 for HCV-OC43; log PFU/ml = 4.83 for HCV-229E and 1.84 for HCV-OC43) versus 10(-3) (HCV-OC43) or 10(-4) (HCV-229E) for reverse transcriptase-polymerase chain reaction amplification (log TCID50/ml = 1.75 for HCV-229E and 1.5 for HCV-OC43; log PFU/ml = 2.3 for HCV-229E and 1.34 for HCV-OC43). There were no false positive signals with other human respiratory pathogens: influenza virus, respiratory syncytial virus and adenovirus. Moreover, each assay was coronavirus serogroup-specific. These results demonstrate the potential usefulness of immunofluorescence with monoclonal antibodies and reverse transcriptase-polymerase chain reaction RNA amplification for the rapid detection of human coronaviruses in infected cell cultures. Both methods could be applied to clinical specimens for the diagnosis of human infections.

In vivo titration of white spot syndrome virus (WSSV) in specific pathogen-free Litopenaeus vannamei by intramuscular and oral routes

“…All shrimp surviving at 120 hpi or longer were not infected with WSSV as determined by these 2 methods. Although it is well established that PCR is more sensitive than IIF (Sizun et al 1998, Walker et al 1998, under these experimental conditions IIF was able to detect WSSV-infected cells in all shrimp detected positive by PCR. In addition, it was very convenient: cheap, easy to perform, yielding rapid results (within 4 h) and with good sensitivity.…”

Molecular diagnosis of respiratory virus infections

“…In a study from Seattle, infections occurred in every month except July and peaked in December of 2003. In a two-year study in Hamilton, Canada, the finding that infections occurred in only one of two years suggested both temporal and geographical Coronavirus infections can be diagnosed by serology, DFA, virus isolation, or NATs, with culture and DFA being the least sensitive, and molecular tests the most sensitive 238,239 . HCoV molecular tests have been described using RT-PCR, NASBA, and LAMP amplification techniques (Table 11).…”

Nucleic acid amplification-based diagnosis of respiratory virus infections

“…CoV infections can be diagnosed by serology or virus isolation, however, culture has proven unreliable as some strains grow poorly [194] and serology has only been used for epidemiological surveys. CoV antigens have been detected in cell culture innoculated with NP or throat wash specimens by standard IF staining with CoVspecific monoclonal antibodies [195].…”