Journal of Medical Virology 31:165-172 (1990) Characterization of a Nucleic Acid Probe for the Diagnosis of Human Coronavirus 2293 Infections S. Myint, D. Harmsen, T. Raabe, and S.G. Siddell MRC Common Cold Unit, Salisbury SP2 8BW, England (S.M.); fnstitut fur Virologie and Immunbiologie der Uniuersitat Wurzburg, 0-8700 Wurzburg, Federal Republic of Germany (D.H., T.R., S.G.S.) A cDNA copy of the HCV229E nucleocapsid pro- tein gene was isolated and characterized. se- quence analysis predicts a nucleocapsid poly- peptide of 389 amino acids with a molecular weight (mol. wt.) of 43,450. Single strand RNA probes derived from the cDNA copy hybridize specifically to HCV229E RNA and approximately 50 pg of intracellular viral RNA can be readily detected. The application of nucleic acid hybrid- ization as a routine procedure for the diagnosis of HCV229E infection is discussed. KEY WORDS: N gene sequence, hybridization analysis, coronavirus INTRODUCTION Human coronaviruses (HCVs) are causative agents of respiratory illness in man. In healthy adults they are associated with common colds of mild to moderate se- verity, usually of 6-8 days duration. The typical symp- toms are nasal catarrh and sore throat, although head- ache, fever, diarrhea, and other symptoms are occasionally reported. Epidemiological data indicate that worldwide HCVs account for 5-35% of all upper respiratory tract infections. HCV infection in children may also lead to lower respiratory illness, including bronchitis and pneumonia. For a review of the biology and pathogenesis of HCVs see Hierholzer and Tannock, 1988.1 HCVs can be divided into two major antigenic groups represented by HCV229E and HCVOC43 Macnaugh- ton et al., 1981; Pedersen et al., 19781. The HCV229E virion is comprised of a positive strand RNA genome, which if HCV is similar to other coronaviruses is about 30 kilobases (kb) in length; a lipid envelope; and three major proteins: the nucleocapsid protein, N (mol. wt. 50,000); the membrane glycoprotein, M (mol. wt. 21,000-25,000); and the spike glycoprotein, S (mol. wt. 186,000) Kemp et al., 1984; Macnaughton and Madge, 1978; Schmidt and Kenny, 19821. Viruses of the OC43 group have an additional surface glycoprotein, the haemagglutinin-esterase, HE (mol. wt. 65 x lo3) Hogue and Brian, 19861. 0 1990 WILEY-LISS, INC. The replication of HCV229E involves the synthesis of a 3 co-terminal set of six subgenomic RNAs Weiss and Leibowitz, 19811. It is assumed that these RNAs are synthesized in the cytoplasm of infected cells by a process of leader-primed discontinuous transcription, as has been described for the murine hepatitis virus, MHV Baric et al., 1985; Makino et al., 1986a; Shieh et al., 19871. Recently, the HCV229E genes encoding the nucleocapsid, membrane, and spike proteins have been cloned and sequenced and their order on the genome has been determined as 5 S-M-N 3 Raabe and Sid- dell, 1989a; Raabe et al., 1990; Schreiber et al., 19891. Raabe et al. 1990 have proposed that the subgenomic RNAs 2, 6,7 function as the mRNAs for the S, M, and N proteins, respectively. Basically, three approaches to the diagnosis of HCV infections have been used. Firstly, there is the isolation and identification of HCVs in cell and organ culture. However, this approach is time consuming and re- quires considerable expertise. Only HCV229E-like vi- ruses can be directly isolated in cell culture and other HCVs have to be isolated in organ culture and, if pos- sible, adapted to cell monolayers see Hierholzer and Tannock, 19881. The second approach is serological. Almost all the standard tests, including serum neutralization, com- plement fixation, and haemagglutinin inhibition (for OC43-like virus) have been used, but they are rela- tively insensitive and are not widely applied. More re- cently, enzyme-linked immunoassays have been devel- oped Kraaijeveld et al., 1980; Macnaughton et al., 1981, 19821 and at the present time they provide the most reliable epidemiological information on HCV in- fections. The major drawback is that paired sera are required and due to the ubiquity of HCV, and the fre- quency of reinfection, the differences in antibody titers between acute and convalescent sera are rarely dra- matic. The third approach to HCV diagnosis involves the direct detection of viral antigens in nasal and pharyn- Accepted for publication February 15, 1990. S. Myints present address is Department of Medical Microbi- ology, University College, London WClE 6JJ, U.K. Address re- print requests there. 166 Myint et al. strand (ds) cDNA was cloned into the Pstl site of the plasmid pAT153 by homopolymeric tailing and used to transform competent E. coli BMH 71-18 cells. A library of transformants containing plasmids with cDNA in- serts of 1-2 kb was selected and individual cDNA in- serts were labelled with 32P by nick translation. The cDNA inserts were then hybridized to Northern blots of poly A RNA from HCV229E infected or uninfected C16 cells. In this w