Genetic and antigenic characterization of recombinant nucleocapsid proteins derived from canine coronavirus and canine respiratory coronavirus in China.

Shuai Lu,Yingzhu Chen,Kun Qin,Jianfang Zhou,Yongliang Lou,Wenjie Tan

doi:10.1007/s11427-016-5038-1

Abstract

To characterize the antigenicity of nucleocapsid proteins (NP) derived from canine coronavirus (CCoV) and canine respiratory coronavirus (CRCoV) in China, the N genes of CCoV (CCoV-BJ70) and CRCoV (CRCoV-BJ202) were cloned from swabs obtained from diseased pet dogs in Beijing and then sequenced. The recombinant NPs (rNPs) were expressed in Escherichia coli and purified by nickel-affinity column and size exclusion chromatography. Sequencing data indicated that the N genes of CCoV-BJ70 and CRCoV-BJ202 belonging to two distinctly different groups were relatively conserved within each subgroup. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that rNPs of CCoV and CRCoV were expressed efficiently and isolated with a final purity of over 95%. Western blot analysis revealed the rNP from CRCoV could cross-react with mice antisera against human coronavirus (HCoV-229E, NL63, OC43, HKU1), while rNP of CCoV had cross-reactivity with only anti-sera against viruses belonging to the same group (HCoV-229E and NL63). In summary, CCoV and CRCoV rNPs were successfully expressed in E. coli and showed antigenic cross-reactivity with antisera raised against human coronaviruses. These findings indicate that further serologic studies on coronavirus infections at the animal-human interface are needed.

Highlights

Coronaviruses are enveloped, positive-stranded RNA viruses that are highly prevalent in animal and human populations (Woo et al, 2009)
Cloning and sequence analysis of nucleocapsid proteins (NP) derived from Canine coronavirus (CCoV) and canine respiratory coronavirus (CRCoV) in China The genes encoding the N proteins of CCoV and CRCoV were amplified and sequenced with the gene lengths being 1,149 bp for CCoV-BJ70 and 1,347 bp for CRCoV-BJ202
Phylogenetic analysis showed that the CCoV N gene obtained in this study had the closest relationship with CCoVNTU336, an Alphacoronavirus strain isolated in Taiwan (Figure 1)

Summary

Introduction

Coronaviruses are enveloped, positive-stranded RNA viruses that are highly prevalent in animal and human populations (Woo et al, 2009). Six types of coronaviruses have been recognized to be pathogenic to humans: HCoV-NL63, HCoV-229E, HCoV-OC43, SARSCoV, HCoV-HKU1 and MERS-CoV. Canine coronavirus (CCoV) and canine respiratory coronavirus (CRCoV) are common viral pathogens responsible for mild to severe enteritis or respiratory symptoms in dog populations (Nicola and Canio, 2008). CCoV infection was first reported in a German military dog with diarrhea in 1971 (Binn et al, 1974). CCoV has been determined to be epidemic globally, and dogs of all breeds and ages appear to be susceptible to infection (Costa et al, 2014; Erles and Brownlie, 2009; Lorusso et al, 2009; Naylor et al, 2002; Yachi and Mochizuki, 2006). In China, CCoV was isolated from the heart and gastrointestinal contents of a sick dog for the first time in 1995 (Kong, 2007), and identified in deceased giant

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