Isolation and characterization of Korean porcine deltacoronavirus strain KNU16-07

Jang, Guehwan; Kim, Seong-Hee; Lee, Yoo Jin; Kim, Seungjoon; Lee, Du Sik; Lee, Kyoung-Ki; Lee, Changhee

The complete genomic sequences of the KNU16-07 isolates at passage 5 (P5) and passage 10 (P10) were determined by performing rapid amplification of the cDNA ends and by using the traditional Sanger method as described previously [6, 7] . The whole genomic nucleotide sequences of KNU16-07-P5 and KNU16-07-P10 were deposited in GenBank under accession numbers MG837130 and MG837131, respectively. The entire structural gene sequences of the isolate at passages 20 and 30 (KNU16-07-P20 and KNU16-07-P30) were also determined by the Sanger method, as described above, and deposited in the GenBank database under accession numbers MG837132 and MG837133, respectively. The PDCoV isolate designated as KNU16-07 was isolated from the feces of a naturally infected piglet from a commercial farm located in Kyungpook Province. KNU16-07 virus produced distinct cytopathic effects (CPE) typical of a PDCoV infection, such as cell rounding, clumping together in clusters, and detachment in infected ST cells at passage 2. In later passages, visible CPE appeared at 12 h post-infection (hpi) and became pronounced by 24 hpi. Virus propagation was confirmed by an immunofluorescence assay using commercial (SD55-197; Medgene Labs, USA) and homemade (KDN4-1) PDCoV N-specific MAbs as described previously [6] . In contrast, no CPE or N-specific staining were evident in mock-inoculated cells (panel A in Fig. 1 ). Ultrastructural analysis of purified virus suspensions revealed multiple virus particles approximately 150 to 200 nm in diameter with typical spike-like surface projections that were morphologically indistinguishable from those of coronaviruses (panel B in Fig. 1) . The viral genome levels in selected passages were examined by quantitative real-time RT-PCR as described previously [6, 11] . The mean cycle threshold (Ct) value was determined to be 15.0, ranging from 13.9 (P10) to 16.1 (P5). The infectious titer of the isolate ranged from 10 7.8 to 10 8.8 TCID50/mL up to P5; this value was stably maintained in later passages. The peak viral titer reached 10 7.8 TCID 50 /mL or more beginning at P5 (panel C in Fig. 1) . Analysis of growth kinetics demonstrated that KNU16-07 replicated rapidly and efficiently in ST cells, reaching a titer ＞ 10 6 TCID 50 /mL by 12 hpi (panel D in Fig. 1) .