The porcine deltacoronavirus accessory protein NS6 is expressed in vivo and incorporated into virions.

Porcine deltacoronavirus (PDCoV) is one of the emerged coronaviruses posing a significant threat to the swine industry. Previous work showed the presence of a viral accessory protein NS6 in PDCoV-infected cells. In this study, we detected the expression of the NS6 protein in small intestinal tissues of PDCoV-infected piglets. In addition, SDS-PAGE and Western blot analysis of sucrose gradient-purified virions showed the presence of a 13-kDa NS6 protein. Further evidences of the presence of NS6 in the PDCoV virions were obtained by immunogold staining of purified virions with anti-NS6 antiserum, and by immunoprecipitation of NS6 from purified virions. Finally, the anti-NS6 antibody was not able to neutralize PDCoV in cultured cells. These data establish for the first time that the accessory protein NS6 is expressed during infection in vivo and incorporated into PDCoV virions.

Successive Passage In Vitro Led to Lower Virulence and Higher Titer of A Variant Porcine Epidemic Diarrhea Virus.

A highly virulent porcine epidemic diarrhea virus (PEDV) appeared in China and spread rapidly to neighbor countries, which have led to great economic losses to the pig industry. In the present study, we isolated a PEDV using Vero cells and serially propagated 100 passages. PEDV SDSX16 was characterized in vitro and in vivo. The viral titers increased to 107.6 TCID50/mL (100th) by serial passages. The spike (S) gene and the whole gene of the SDSX16 virus was fully sequenced to assess the genetic stability and relatedness to previously identified PEDV. Along with successive passage in vitro, there were 18 nucleotides (nt) deletion occurred in the spike (S) gene resulting in a deletion of six amino acids when the SDSX16 strain was passaged to the 64th generation, and this deletion was stable until the P100. However, the ORF1a/b, M, N, E, and ORF3 genes had only a few point mutations in amino acids and no deletions. According to growth kinetics experiments, the SDSX16 deletion strain significantly enhanced its replication in Vero cells since it was passaged to the 64th generation. The animal studies showed that PEDV SDSX16-P10 caused more severe diarrhea and vomiting, fecal shedding, and acute atrophic enteritis than SDSX16-P75, indicating that SDSX16-P10 is enteropathogenic in the natural host, and the pathogenicity of SDSX16 decreased with successive passage in vitro. However, SDSX16-P10 was found to cause lower levels of cytokine expression than SDSX16-P75 using real-time PCR and flow cytometry, such as IL1ß, IL6, IFN-ß, TNF-α, indicating that SDSX16-P10 might inhibit the expression of cytokines. Our data indicated that successive passage in vitro resulted in virulent attenuation in vivo of the PEDV variant strain SDSX16.