Molecular characterization of a new species in the genus Alphacoronavirus associated with mink epizootic catarrhal gastroenteritis

Abstract

A coronavirus (CoV) previously shown to be associated with catarrhal gastroenteritis in mink (Mustela vison) was identified by electron microscopy in mink faeces from two fur farms in Wisconsin and Minnesota in 1998. A pan-coronavirus and a genus-specific RT-PCR assay were used initially to demonstrate that the newly discovered mink CoVs (MCoVs) were members of the genus Alphacoronavirus. Subsequently, using a random RT-PCR approach, full-genomic sequences were generated that further confirmed that, phylogenetically, the MCoVs belonged to the genus Alphacoronavirus, with closest relatedness to the recently identified but only partially sequenced (fragments of the polymerase, and full-length spike, 3c, envelope, nucleoprotein, membrane, 3x and 7b genes) ferret enteric coronavirus (FRECV) and ferret systemic coronavirus (FRSCV). The molecular data presented in this study provide the first genetic evidence for a new coronavirus associated with epizootic catarrhal gastroenteritis outbreaks in mink and demonstrate that MCoVs possess high genomic variability and relatively low overall nucleotide sequence identities (91.7 %) between contemporary strains. Additionally, the new MCoVs appeared to be phylogenetically distant from human (229E and NL63) and other alphacoronaviruses and did not belong to the species Alphacoronavirus 1. It is proposed that, together with the partially sequenced FRECV and FRSCV, they comprise a new species within the genus Alphacoronavirus.

Fig. 1.

Neighbour-joining tree of coronaviruses based on full genomic sequences. The tree was inferred using mega4. Bootstrap support values >95 % are indicated. Previously defined genera and species and a potential new species (Alphacoronavirus 2) are delineated by the bars on the right. The naming of these genera is as described in the 2009 report of the ICTV. Bar, number of nucleotide substitutions per site. TCoV, Turkey coronavirus; see text and tables for other abbreviations.

Fig. 2.

Neighbour-joining tree of coronaviruses based on N gene sequences. The tree was inferred using mega4. Bootstrap support values ≥90 % are indicated for every node except for that between alphacoronaviruses. Bar, number of nucleotide substitutions per site. See Fig. 1 for abbreviations.

Fig. 3.

Schematic diagram of the gene arrangements of the 3′-terminal region of the MCoV, FRECV, FCoV, CCoV and TGEV genomes. The ORFs coding for structural (S, E, M and N) and non-structural (3a/3b/3c, 3x, 7a and 7b) proteins are represented in boxes. Dotted lines represent genomic regions that have not yet been sequenced.