Detection and characterization of bovine-like coronaviruses from four species of zoo ruminants

1. Introduction

Coronaviruses (CoVs) are a member of the order Nidovirales and family Coronaviridae, each member of which contains a single-stranded, non-segmented RNA positive-sense genome that is 26–31 kb in size. CoVs are classified into three different genetic and antigenic genera, each of which is divided into several subgenera. Bovine-like CoVs and bovine CoVs (BCoVs), which belong to Betacoronavirus 1, contain five major structural proteins: nucleocapsid (N), transmembrane (M), spike (S), small membrane (E), and hemagglutinin/esterase (HE) (Lai and Cavanagh, 1997). Betacoronavirus 1 also include the human CoVs-OC43 (HCoV-OC43), porcine hemagglutinating encephalomyelitis virus (PHEV), equine coronavirus (ECoV), canine respiratory coronavirus (CRCoV) and bubaline coronavirus (BuCoV) (Decaro et al., 2008, Erles et al., 2007, Guy et al., 2000, Haring and Perlman, 2001, Vijgen et al., 2006).

Bovine-like CoVs were first reported in ruminant species in the late 1970s (Tzipori et al., 1978). Later, bovine-like CoVs were also identified in wild animals (sitatunga, waterbuck, musk oxen, llamas, alpacas, white-tailed deer, giraffe, Sambar deer, and sable antelope) (Cebra et al., 2003, Chasey et al., 1984, Hasoksuz et al., 2007, Tsumemitsu et al., 1995). BcoVs cause newborn calf diarrhea (CD), winter dysentery (WD) in adult cattle, and respiratory tract illness in calves and feedlot cattle (Lathrop et al., 2000, Saif et al., 1991, Snodgrass et al., 1986). BcoVs also infect the respiratory tract of cattle and cause disease therein (Cho et al., 2001, Hasoksuz et al., 1999).

The genome of Betacoronavirus 1 encodes a short, spike-like protein known as HE. BcoV uses N-acetyl-9-O-acetylneuraminic acid (sialic acid) as a receptor to initiate infection (Schultze and Herrler, 1992). Although S protein has an affinity for 9-O-acetylated sialic acid, HE protein has been identified as the principal BcoV sialic acid-binding protein (Schultze et al., 1991a, Schultze et al., 1991b). Therefore, HE may be involved in virus entry and/or virus release from infected cells (Schultze et al., 1991a). The S protein, which is composed of two subunits, S1 and S2, has several important functions during the virus–host interaction. The S1 subunit is associated with binding to host cell receptors, whereas the S2 subunit is the viral M protein that mediates the fusion of viral and cellular membranes. Thus, S protein is critical for virus entry and pathogenesis (Schultze et al., 1991b). The variation in host range and tissue tropism among CoV is largely attributable to variations in the S protein (Gallagher and Buchmeier, 2001).

Several Korean BcoV strains have recently been characterized at the molecular level (Jeong et al., 2005a, Jeong et al., 2005b, Park et al., 2006). However, such analyses of bovine-like CoV strains circulating in Korea have not yet been conducted. In this study, we report sequence and phylogenetic analyses of the S and HE protein genes of five bovine-like CoVs isolated from four species of zoo ruminants.

2. Materials and methods

3. Results and discussion

Of the 12 fecal samples tested, five (one wisent, two Himalayan tahr, one sitatunga, and one nyala) were positive for CoV. Five fecal samples were used for virus isolation and sequencing. All of the S genes sequenced contained an open reading frame (ORF) of 4092 nucleotides, encoding a predicted protein of 1363 amino acid (aa) residues and molecular weight of approximately 150 kDa. Sequence analysis of the five identified strains showed a 99.9% amino acid identity and a genetic relatedness of 99.4–99.5% to two other Korean isolates, BcoV-0501 and BcoV-0502. Thus, the most recent isolates, BcoV-0501 and BcoV-0502 (identified in 2005), had the highest nucleotide identity with the five bovine-like CoVs, whereas they showed only 97.8–97.9% identities with the BC94/1994 (DQ389641) and Sun5/1994 (DQ389657) strains. In total, 86 nucleotide substitutions were found compared to the Mebus strain, but no insertions or deletions were detected in any sequence. Also, 34 substitutions were found in the complete aa sequence of the viral glycoprotein S.

A phylogenetic analysis using reference strains from all known groups of CoVs demonstrated that the bovine-like CoVs isolated in this study belonged to Betacoronavirus 1 clustering with Korean BcoVs isolates collected after 2004 (Fig. 1a). The bovine-like CoVs isolated were closely related to two Korean strains (BcoV-0501 and BcoV-0502) identified in 2005, suggesting that this virus may have been transmitted from cattle to zoo ruminants. Korean BcoVs strains collected before 2004 were divided into several clusters. A BcoV-white-tailed deer strain isolated in 1994 (Tsumemitsu et al., 1995) formed a cluster together with Korean BcoV-KWD1–10 isolates, with the exception of KWD 7.

All of the HE genes contained an ORF of 1272 nucleotides, encoding a predicted protein of 424 aa residues and molecular weight of approximately 47.6 kDa. In total, 27 nucleotide substitutions were found compared to the Mebus strain, but no insertions or deletions were detected in any sequence. Also, five substitutions were found in the complete aa sequence of the S glycoprotein.

Phylogenetic analyses were carried out to determine genetic relationships based on the nucleotide sequences of the HE gene (Fig. 1b). The Korean BcoV strains were divided into three groups. The first group consisted mainly of all of the Korean isolates. CoV strains from zoo ruminants were grouped within a cluster of other Korean CoV strains isolated after 2004. The CoV strains clustered with the BcoV-0501/2005 strain, which was identified in 2005. The second group included some Korean strains isolated in 2002 and bovine-like strains from wild ruminants. The last group consisted mainly of BcoV vaccine strains.

In this study, we compared the S and HE genes of Korean bovine-like CoVs isolated directly from feces of zoo ruminants to determine their relatedness to bovine-like CoVs from wild ruminants at other locations. However, no relationship between Korean bovine-like CoVs and those from wild ruminants at other locations was detected. Sequence analysis of the S and HE genes, however, indicated that the strains show a high similarity to BcoV. This was confirmed by phylogenetic analysis, thus supporting data obtained previously (Alekseev et al., 2008). The CoV strain isolated in this study appears to derive from the same ancestor as the BcoV 05/01 and BcoV 05/02 strains isolated from cattle in Korea. Also, the sequences of the S and HE genes of Korean CoV strains represented on the tree grouped according to year of isolation. These data support the hypothesis that genetic difference may be related more to the time of the appearance of an outbreak than to its geographic location (Alekseev et al., 2008, Jeong et al., 2005b).

The 99.9% aa identity of the five bovine-like CoV strains suggests that the zoo ruminants were infected by the same bovine-like CoV. The high genetic similarity with bovine-like CoVs from the zoo ruminants and Korean BcoV strains was also confirmed, suggesting that cattle may be potential reservoirs for CoVs that infect zoo ruminants. However, additional isolation and characterization of bovine-like CoVs from other heterologous hosts are required to clarify the origin of bovine-like CoVs and their relationship to other BcoVs.

Since the 1970s, bovine-like CoVs have been recognized as being distributed worldwide, with many reports indicating that the virus had been detected and/or isolated in the United States, Europe, and Asia. BcoVs have been detected in the feces of calves and adult cattle with diarrhea in Korea (Jeong et al., 2005a, Jeong et al., 2005b, Park et al., 2006). In addition, antibodies to BcoV have been demonstrated in Korean native goats (Capra hircus), and 1.0% of Korean native goats were serologically positive for BcoV (Yang et al., 2008). However, this to our knowledge is the only report of detection and characterization of bovine-like CoVs in Korea that has been published to date. No specific genomic characteristics that could account for the host specificity exhibited by bovine-like CoVs were identified by comparative sequence analysis.