Comparative sequence analysis of the distal one-third of the genomes of a systemic and an enteric ferret coronavirus

Abstract

Ferret systemic coronavirus (FRSCV) infection is associated with an emerging, highly fatal disease of ferrets. Enhanced macrophage tropism and the resulting induction of pyogranulomatous lesions are shared with feline infectious peritonitis virus (FIPV) infection in cats, but are not features of ferret enteric coronavirus (FRECV) infection. Comparative sequence analysis of the distal one-third of the genomes of one FRSCV and one FRECV strain showed that these two ferret coronaviruses share >96% nucleotide sequence identities in the membrane (M), nucleocapsid (N) and non-structural protein genes (partial polymerase, open reading frames [ORFs] 3 and 7b). The envelope (E) protein gene showed a moderate nucleotide sequence similarity of 91.6%. In contrast, nucleotide and amino acid sequence similarities observed with the spike (S) protein were only 79.5 and 79.6%, respectively. Twenty-one amino acid differences within a 195-199-amino acid C-terminal portion of the S protein were conserved between 3 strains each of FRSCV and FRECV. Both systemic and enteric strains were found to carry a single ORF 3 gene with truncated proteins observed in two out of three FRSCV strains examined. The two enteric strains analyzed each contained an intact ORF 3 gene. Phylogenetically, FRSCV is more closely related to FRECV than to other group 1 coronaviruses.

Fig. 1

Alignment of the deduced partial amino acid sequences of the spike proteins of FRSCV MSU-1, FRSCV MSU-S, FRSCV WADL, FRECV MSU-2, FRECV-MSU1 and FRECV 1202. Residues that match the majority sequence are shown as dots.

Fig. 2

Alignment of the genotype-specific diagnostic PCR target regions within the spike genes of FRSCV MSU-1 and FRECV MSU-2. Matching nucleotides are shown as dots. The broken line indicates a deletion. Primer sequences are underlined.

Fig. 3

Alignment of the deduced amino acid sequences of the spike proteins of FRSCV MSU-1 and FRECV MSU-2. Conserved residues are shown as dots.

Fig. 4

Alignment of the ORF 3 region sequences of FRECV MSU-2 and FRSCV MSU-1. Matching nucleotides are shown as dots. Deletions are represented by broken lines. ATG and TAG or TAA codons are underlined.

Fig. 5

Phylogenetic trees based on deduced partial amino acid sequences of the: (A) partial polymerase, (B) spike, (C) envelope, (D) membrane, and (E) nucleocapsid proteins of FRSCV MSU-1, FRECV MSU-2 and representative strains of group 1 coronaviruses. SARS virus was included as the outgroup sequence. Bootstrap values are indicated at the nodes.

Fig. 6

S-gene genotype-specific diagnostic RT-PCR on clinical samples. (A) Genotype 1-specific assay, 157-bp positive amplicon observed only on Lanes 2–6. (B) Genotype 2-specific assay, 147-bp positive amplicon observed only on Lanes 7–8. Lane designations for both panels A and B: Lanes 1 and 10, 100-bp DNA ladder; Lane 2, FRSCV MSU-1 positive lung; Lane 3, FRSCV MSU-1 positive kidney; Lane 4, FRSCV WADL positive lymph node; Lane 5, FRSCV MSU-S positive spleen; Lane 6, FRSCV MSU-S positive intestine; Lane 7, FRECV MSU-2 positive feces; Lane 8, FRECV 1202 positive feces; Lane 9, negative control (sterile water).