Attenuation of an original US porcine epidemic diarrhea virus strain PC22A via serial cell culture passage

Abstract

Although porcine epidemic diarrhea (PED) has caused huge economic losses in the pork industry worldwide, an effective live, attenuated vaccine is lacking. In this study, an original US, highly virulent PED virus (PEDV) strain PC22A was serially passaged in Vero CCL81 and Vero BI cells. The virus growth kinetics in cell culture, virulence in neonatal pigs and the whole genomic sequences of selected passages were examined. Increased virus titers and sizes of syncytia were observed at the 65th passage level (P65) and P120, respectively. Based on the severity of clinical signs, histopathological lesions and the distribution of PEDV antigens in the gut, the virulence of P100 and above, but not P95C13 (CCL81), was markedly reduced in 4-day-old, caesarian-derived, colostrum-deprived piglets. Subsequently, the attenuation of P120 and P160 was confirmed in 4-day-old, conventional suckling piglets. Compared with P120, P160 replicated less efficiently in the intestine of pigs and induced a lower rate of protection after challenge. Sequence analysis revealed that the virulent viruses [P3 and P95C13 (CCL81)] had one, one, sixteen (including an early termination of nine amino acids) and two amino acid differences in non-structure protein 1 (nsp1), nsp4, spike and membrane proteins, respectively, from the fully attenuated P160. However, the overall pattern of attenuation-related genetic changes in PC22A differed from those of the other four pairs of PEDV wild type strains and their attenuated derivatives. These results suggest that PEDV attenuation can occur through multiple molecular mechanisms. The knowledge provides insights into potential molecular mechanisms of PEDV attenuation.

Attenuation and characterization of porcine enteric alphacoronavirus strain GDS04 via serial cell passage

“…The mortality of newborn piglets in each group was recorded from 1 to 9 d.p.i.. Xu, et al Veterinary Microbiology 239 (2019) 108489 significantly higher than those of P15 and P67 at 24 hpi, similar to observations in attenuated PEDV vaccine (Lin et al, 2017), indicating that P100 had better adaptation in cells in vitro. Usually, adaptation in cell lines coincides with attenuation of the viruses, such as African swine fever virus (ASFV) isolate Georgioa and PEDV isolate PC22A, in swine Lin et al, 2017). The higher fitness of P100 in Vero cells might suggest the changed pathogenicity of PEAV.…”

“…Pathogenic microorganisms serially passaged in cells (Jie et al, 2018) is one of the most common methods to prepare live-attenuated vaccine. Vero cells as a stable African green monkey kidney cell line (Rhim et al, 1969) is commonly used to attenuate CoVs like PEDV (Lin et al, 2017). In this study, PEAV GDS04 was passaged regularly to a total of 100 passages in Vero cells and the growth rate of P15, P67 and P100 was determined in vitro.…”

“…Serial virus passaging and plaque-purification in Vero cells were performed as previously described with some modifications (Lin et al, 2017;Xu et al, 2019a). Briefly, Vero cells were cultured in 6-well plates, and washed three times with sterile pH 7.4 1 × phosphate buffer saline (PBS) when 90% confluent.…”

“…Kinetics of PEAV replication in Vero cells were performed as previously described with some modifications (Lin et al, 2017). Briefly, Vero cell monolayer was inoculated with P15, P67 and P100 at a MOI = 0.1.…”

“…These cell-passaged strains are then tested in animals to determine whether they have less virulence and higher immunogenicity before being used to prepare live-attenuated vaccines. Of note, several groups have used cell passage to prepare porcine CoVs attenuated strains, such as PEDV (Lin et al, 2017) and TGEV (Motovski et al, 1985), which laid a good foundation for preparation of live-attenuated vaccines.…”