Mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo

Abstract

Previously, we have reported that a serial passage of 83P-5 strain of porcine epidemic diarrhea virus (PEDV) in Vero cells resulted in a growth adaptation of the virus in cultured cells at the 22nd passage. In this study, we further maintained the 83P-5 in Vero cells up to the 100th passage and analyzed changes in the spike (S), membrane (M), and nucleocapsid (N) gene sequences and pathogenicity of the virus at the 34th, 61st, and 100th passage levels. Sequence analyses revealed a strong selection for the S gene of 83P-5 in Vero cells, and virtually all mutations occurring at the 34th and 61st passages had been carried over to the 100th-passaged virus. In contrast, the viral M and N genes showed a strong conservation during the serial passage. Pigs experimentally infected with the 34th- or 61st-passaged virus, but not the 100th-passaged virus, exhibited diarrhea, indicating an attenuation of the 83P-5 at the 100th passage. Interestingly, S protein of the attenuated 100th-passaged 83P-5 showed a remarkable sequence similarity to that of previously reported DR-13 strain of attenuated PEDV that also had been established by serial passage in Vero cells. Further studies will be required to define whether the mutations in the S gene of 83P-5 that had been selected and accumulated during the serial passages are indeed the causalities of the growth adaptation in vitro and the attenuation of virulence in vivo.

Fig. 1

Schematic representation of mutations in the S protein of the parent, 34th-, 61st-, and 100th-passaged 83P-5. Signal peptide (SP), transmembrane domain (TM), and neutralizing epitopes (SS2, SS6, and 2C10) are shown as gray boxes. Amino acid numbering corresponds to the parent virus. Dash indicates deletion

Fig. 2

Alignment of the deduced amino acid sequences of the S protein of the parent and passaged PEDV 83P-5 strains with that of the parent and attenuated DR13 strains [27]. Dots represent amino acids that are identical to those in the parent 83P-5. Potential N-linked glycosylation sites of N-{P}-[ST] are underlined. Boxes indicate the signal peptide (position 1–21), transmembrane domain (1334–1356), and neutralizing epitopes (748–755; SS2, 764–771; SS6 and 1368-1374; 2C10)

Fig. 3

Phylogenetic analysis of the full-length S gene sequence of PEDV. Neighborhood-joining tree was generated with 1,000 bootstrap replications using Clustal X v1.83 [39] and viewed by MEGA v4.0.2. The phylogenetic tree was rooted with an out-group TGEV strain (accession number 811785). GenBank accession numbers of reference PEDVs for the S gene are AY167585 (Chinju99), AF353511 (CV777), DQ462404 (DR13 attenuated), DQ862099 (DR13 parent), EU031893 (DX), AY653204 (JS-2004-2), AB548622 (Kawahira), GU180142 (KNU-0801), GU180143 (KNU-0802), GU180144 (KNU-0901), GU180145 (KNU-0902), GU180146 (KNU-0903), GU180147 (KNU-0904), GU190148 (KNU-0905), DQ985739 (LJB/03), EF185992 (LZC), AB548624 (MK), AB548623 (NK), and AF500215 (Spk1)