Determination of Ferret Enteric Coronavirus Genome in Laboratory Ferrets

Ferret enteric coronavirus (FRECV) RNA was detected in laboratory ferrets. Analysis of the complete genome sequence of 2 strains, FRCoV4370 and FRCoV063, revealed that FRECV shared 49.9%–68.9% nucleotide sequence identity with known coronaviruses. These results suggest that FRECV might be classified as a new species in the genus Alphacoronavirus.

Ferret enteric coronavirus (FRECV) RNA was detected in laboratory ferrets. Analysis of the complete genome sequence of 2 strains, FRCoV4370 and FRCoV063, revealed that FRECV shared 49.9%-68.9% nucleotide sequence identity with known coronaviruses. These results suggest that FRECV might be classified as a new species in the genus Alphacoronavirus.  (2,3). Genetic analyses based on these partial sequences showed that FRCoV is closer to mink coronavirus (MCoV) than to other CoVs and appears to be a member of the genus Alphacoronavirus in the subfamily Coronovirinae, which also contains the genera Betacoronavirus and Gammacoronavirus (4). To further understand the constellation of FRCoVs, we analyzed the complete genome.

The Study
In our previous study, we detected ferret hepatitis E virus (HEV) in fecal samples from laboratory ferrets and confirmed that 40 (63.5%) of the 63 ferrets were infected with ferret HEV (5). For the observation of the ferret HEV particles by electron microscopy, we used a 10% suspension prepared from the fecal specimen from 1 of the ferrets (no. F4370), concentrated by ultracentrifugation and purified by sucrose gradient ultracentrifugation. However, instead of finding ferret HEV particles, we observed many coronavirus-like particles in fractions 4, 5, and 6, with densities of 1.230 g/cm 3 , 1.214 g/cm 3 , and 1.198 g/cm 3 (data not shown). These particles ranged from 60 nm to 120 nm in diameter; most of them had a spike structure, suggesting that the ferrets were infected with a CoV-like virus.
RT-PCR results revealed that, of the 63 ferret fecal specimens, 22 (34.9%) were positive for FRSCV RNA, 53 (84.1%) were positive for FRECV RNA, and 15 (23.8%) were positive for both FRSCV and FRECV RNA. The specimen from ferret F4370 was positive only for FRECV RNA. These results indicated that the ferrets were infected extensively with FRCoV. However, we observed no signs such as weight loss or diarrhea in the ferrets.
We extracted RNA from a pool of fractions 4, 5, and 6 from the F4370 fecal sample suspension and analyzed the complete genome sequence of FRCoV by using a next-generation sequence analysis (6). FRCoV4370 (GenBank accession no. LC119077) has a genome size, gene order, genomic organization, and structure similar to those of known alphacoronaviruses. The complete genome of FRCoV4370 contains 28,525 nt and a poly (A) tail. Except for the 5′-terminus 261 nt and 3′-terminus 247 nt untranslated regions, FRCoV4370 encodes 9 proteins: ORF1a, ORF1a/1b, S, 3c, E, M, N, 3x, and 7b (Table). Although the ORF7a protein was found in the MCoVs WD1127 (GenBank accession no. HM245925) and WD1133 (accession no. HM245926), we did not find it in FRCoV4370.
The complete genome of FRCoV4370 shared 49.9%-68.9% nucleotide identity with other known CoVs.

Determination of Ferret Enteric Coronavirus Genome in Laboratory Ferrets
Author Phylogenetic trees based on the complete genome demonstrated that FRCoV4370 is closer to MCoVs than to other CoVs, although it clearly separated into a distinct cluster ( Figure, panel A). We observed similar phylogenetic clustering when we compared ≈8,300 nt sequences of the 3′-termini of CoVs. FRCoV4370 forms a new cluster with FRSCV MSU-1, FRECV MSU-2, and No22, being separated from the cluster formed by MCoVs (Figure, panel B). In addition, BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the nucleotide sequences described previously revealed that FRCoV4370 shared 94% identity with FRECV MSU-2, which is higher than that of FRSCV MSU-1 (89%), indicating that FRCoV4370 belongs to the FRECVs.
As is the case for other CoVs, 2 long ORFs were predicted in the FRCoV4370 genome: ORF1a contains 11,967 nt, from nt 262 to 12228, encoding 3,988 aa; ORF1b encodes 7,830 nt, from nt 12393 to 20222. The coronaviruses have a pseudoknot tertiary structure that allows a ribosomal shift of the reading frame between ORF1a and ORF1b (7-9). We also found the slippery sequence for the ribosomal shift (UUUAAAC) in the FRCoV4370 genome at nt positions 12192-12198. The ribosomal shift may have resulted in generation of the ORF1a/b protein encoding 19,962 nt and the deduced 6,653 aa.
We identified 4 structural proteins in FRCoV4370: S, E, M, and N. The S protein is the largest, consisting of 1,449 aa, which shared 92.5% aa identity with FRECV MSU-2, 81.4% with FRSCV MSU-1, and 84.5% with FRECV No22. The BLAST analysis of the S protein showed that it had low aa identities (43%-66%) with other CoVs.  Because the S protein is a major inducer of virus-neutralizing antibodies, the antigenicity and serotype of FRCoVs might be different from those of other CoVs. The E protein, the smallest structural protein, encodes 82 aa (which is the same number encoded by other known FRCoVs) and shared 94.8% aa identity with FRECV MSU-2, 85.6% with FRSCV MSU-1, and 86.7% with FRECV No22. The 263-aa M protein shared 81.6% aa identity with FRECV MSU-2. The low aa identity between the FRECV strains suggests that the membrane gene is highly variable. The N protein of FRCoV4370 contains 374 aa, making it the shortest among the alphacoronaviruses. The N protein shared 96.3% aa identity with FRECV MSU-2, 93.4% with FRSCV MSU-1, and 80.2% with FRECV No22.
ORF3c was identified in FRCoV4370, as observed in MCoVs. This protein is an accessory triple-spanning membrane protein and is analogous to SARS-CoV 3a protein (10). The predicted ORF3c protein contains 247 aa and shared 96.8% aa identity with FRECV MSU-2 and 77.6% with FRECV No22. The 2 other nonstructural proteins, 3x and 7b, are located downstream of the N protein and encode 74 aa and 204 aa, respectively.
We also detected FRECV RNA in the fecal specimens of 9 ferrets from another farm in the United States and analyzed the complete genome of 1 strain, FRCoV063 (LC215971). The genome of FRCoV063 shared 94.0% nucleotide identity with FRCoV4370 and had a similar structure (Table; Figure), suggesting that ferret coronavirus infection is common in ferrets, and genetically similar FRCoV strains circulate at the ferret farms.

Conclusions
Since the initial identification of FRCoV in ferrets in 2006, many sequences of FRSCV and FRECV strains have been analyzed (1)(2)(3)(11)(12)(13)(14)(15). However, the complete genome sequences have not been determined and added to the public databases. In this study, we successfully analyzed the complete genome of 2 strains of FRECV, FRCoV4370 and FRCoV063, and found that they shared 94.0% nucleotide identity with each other but 49.9%-68.9% nucleotide identities with other known CoVs, suggesting that the ferret coronaviruses might be classified as a new species in the genus Alphacoronavirus. This new knowledge of the complete genome sequence of FRECV will contribute to investigations of the diversity of animal CoVs and will help establish new taxonomic units. This study was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, and Science, Japan.
Dr. Li is a senior researcher at the National Institute of Infectious Diseases in Tokyo, Japan. His research focuses on epidemiology, the expression of viral proteins, and advances in the diagnosis of and vaccines for hepatitis E virus.