Phylogenetic analysis of a highly conserved region of the polymerase gene from 11 coronaviruses and development of a consensus polymerase chain reaction assay

Abstract

Viruses in the genus Coronavirus are currently placed in three groups based on antigenic cross-reactivity and sequence analysis of structural protein genes. Consensus polymerase chain reaction (PCR) primers were used to obtain cDNA, then cloned and sequenced a highly conserved 922 nucleotide region in open reading frame (ORF) 1b of the polymerase (pol) gene from eight coronaviruses. These sequences were compared with published sequences for three additional coronaviruses. In this comparison, it was found that nucleotide substitution frequencies (per 100 nucleotides) varied from 46.40 to 50.13 when viruses were compared among the traditional coronavirus groups and, with one exception (the human coronavirus (HCV) 229E), varied from 2.54 to 15.89 when compared within these groups. (The substitution frequency for 229E, as compared to other members of the same group, varied from 35.37 to 35.72.) Phylogenetic analysis of these pol gene sequences resulted in groupings which correspond closely with the previously described groupings, including recent data which places the two avian coronaviruses--infectious bronchitis virus (IBV) of chickens and turkey coronavirus (TCV)--in the same group [Guy, J.S., Barnes, H.J., Smith L.G., Breslin, J., 1997. Avian Dis. 41:583-590]. A single pair of degenerate primers was identified which amplify a 251 bp region from coronaviruses of all three groups using the same reaction conditions. This consensus PCR assay for the genus Coronavirus may be useful in identifying as yet unknown coronaviruses.

Fig. 1

Deduced amino acid sequence of the polymerase motif region from open reading frame (ORF) 1b of the pol gene of 11 coronaviruses. The mouse hepatitis virus (MHV), infectious bronchitis virus (IBV) and 229E sequences are derived from published sequences (see text). The first amino acid in this figure corresponds to amino acid 466 of the IBV ORF 1b (Lee et al., 1991). Amino acids 79 through 307 correspond to 228 of the 258 amino acids representing the conserved polymerase motif common to coronaviruses, toroviruses and arteriviruses (see Fig. 5 in den Boon et al., 1991). The highly conserved SDD or GDD polymerase motif (Poch et al., 1989) is identified by asterisks. Capitalized letters indicate amino acids which are conserved in all 11 sequences.

Fig. 2

Unrooted dendogram showing Kimura’s distances (represented by branch lengths) for cDNA sequences from a 922 nucleotide region of open reading frame (ORF) 1b of the pol gene of 11 coronaviruses (see text for details). Numbers represent the results of a bootstrap analysis and indicate the number of times out of 100 iterations that these branch points were identified. Sequence for the eight coronavirus sequences reported here is available from GenBank under the following accession numbers: bovine coronavirus (BCV), AF124985; canine coronavirus (CCV), AF124986; feline infectious peritonitis virus (FIPV), AF124987; hemagglutinating encephalomyelitis virus of swine (HEV), AF124988; OC43, AF124989; sialodacryoadenitis virus of rats (SDAV), AF124990; turkey coronavirus (TCV), AF124991; transmissible gastroenteritis virus (TGEV), AF124992.

Fig. 3

cDNA sequences for a subregion of the 922 nucleotides from open reading frame (ORF) 1b of the pol gene used for the analysis shown in Fig. 2. (Nucleotide number 1 of this 922 nucleotide-long region corresponds to nucleotide number 13 853 in the infectious bronchitis virus (IBV) pol sequence (Boursnell et al., 1987). This Figure shows nucleotides number 101 through 400. The regions targeted by the two degenerate primers (CV2Bp and CV4Bm, see text for sequence) used in the consensus polymerase chain reaction (PCR) assay for the genus Coronavirus are underlined.

Fig. 4

Polymerase chain reaction (PCR) products for ten coronaviruses [OC43, bovine coronavirus (BCV), mouse hepatitis virus (MHV), sialodacryoadenitis virus of rats (SDAV), 229E, feline infectious peritonitis virus (FIPV), transmissible gastroenteritis virus (TGEV), canine coronavirus (CCV), infectious bronchitis virus (IBV), turkey coronavirus (TCV)] using the consesus PCR primers (2Bp and 4Bm, see text for sequence) for the genus Coronavirus. Twenty μl of reaction product were run on a 4% agarose gel (NuSieve 3:1, FMC BioProducts, Rockland, ME) and stained with 1 μg/ml ethidium bromide. Also included on the gel were: reaction product from PCR using 1 pg of plasmid containing target sequence from human coronavirus (HCV)-OC43 as positive control (pOC43); reaction products from negative control samples (water only) which were carried through both the reverse transcriptase (RT) and PCR steps (RT neg) or the PCR step alone (PCR neg); 1 μg of 123 bp molecular size standards (Bethesda Research Labs, Bethesda, MD).