Vero cells were inoculated with FJzz1 variants F20, F50, F100, F150 or F200 at an MOI of 0.01 to analyze the changes in their biological characteristics during the serial passage in vitro. A CPE, characterized by multiple regional cell fusion and syncytium formation, was observed at 10 hpi in the cells inoculated with high-passage variants such as F150 and F200. By contrast, Vero cells inoculated low-passage variants, especially F20, developed visible CPE at 15 hpi, 5 h later than the high-passage variants (Fig. 1A). Vero cells infected with F20, F50, F100, F150, or F200 displayed specific green fluorescence when treated with a Mab directed against PEDV N protein, but no green fluorescence was observed in the uninfected cells (Fig. 1B). All five passages of FJzz1 variants developed visible round plaques on confluent Vero cells in six-well plates. It is noteworthy that the high-passage FJzz1 variants showed higher proliferation than the low-passage variants, which contributed to larger size form of plaques (0.21–0.26 mm) in Vero cells infected with the high-passage FJzz1 variants than those with the low-passage variants (0.08–0.12 mm) (Fig. 1C). The multistep growth kinetics of F20, F50, F100, F150, and F200 based on the TCID50 at different hpi were visualized, and showed that the viral titers of the low-passage variants (F20, F50, and F100) peaked at 30 hpi, whereas those of the high-passage variants (F150 and F200) peaked at 24 hpi, 6 h before the low-passage variants (Fig. 1D). In conclusion, these results indicate that the sensitivity and adaptability of the FJzz1 variants to Vero cells gradually increased during their serial passage in vitro.
Figure 1. Biological characterization of FJzz1 variants during serial passage in vitro. A Vero cells were mock infected or infected with FJzz1 variants F20, F50, F100, F150, or F200 at an MOI of 0.01, and a CPE was observed at 24 hpi. Scale bar = 100 µm. B Monolayers of Vero cells inoculated with FJzz1 variants F20, F50, F100, F150, or F200 were tested with an IFA using a MAb to PEDV N protein. Scale bar = 50 µm. C Crystal-violet-stained plaques formed on the monolayers of Vero cells inoculated with FJzz1 variants at different passages at 3 dpi. D Vero cells were infected with FJzz1 variants at different passages at an MOI of 0.01. The cell lysates were collected at the designated times and titrated with a TCID50 infectivity assay. Asterisk (*) indicates a significant difference between FJzz1-F20 and FJzz1-F200 (*P < 0.05; **P < 0.01; ***P < 0.001).
To further analyze the genetic variation in the FJzz1 variants, the full-length genomic sequences of these variants were aligned using Clustal W in MegAlign, and the results showed that relative to F20, the other passaged variants (F50, F100, F150, and F200) had 7, 14, 18 and 19 aa changes, respectively (Table 1), indicating that genomic sequences of the FJzz1 variants tended to be stable during serial passage. Different degrees of aa changes were detected in the ORFs of PEDV, including ORF1a, ORF1b, S, ORF3 and M. F100, F150 and F200 showed the same numbers of aa changes in Nsp2, ORF3 and M, which accounted for 0.13%, 0.44% and 0.44% of their total aa, respectively. The change rate of the S protein in FJzz1 variants increased during their serial propagations in vitro, reaching the peak change rate (1.01%) in F200. Notably, the aa changes in FJzz1 variants were mainly concentrated in the S protein, with high change rates ranging from 72.22% to 85.71% of the total changed aa (Fig. 2A), whereas other proteins (Nsp2, Nsp3, Nsp13, ORF and M) had lower aa change rates. The changed aa of the S protein in the FJzz1 variants were scattered in both the S1 and S2 subunits, which is consistent with the differences observed between the classical strain and its attenuated variants and between other mutant epidemic strains and their attenuated variants (Fig. 2B). In addition, a phylogenetic tree was constructed based on the S gene of the FJzz1 strain and another 60 reference strains for which the complete S gene sequences were available in GenBank. The phylogenetic analysis showed that the FJzz1 strain and the 60 reference strains formed two genogroups: the G1 genotype mainly contained the classical strains, such as CV777 and DR13, whereas the G2 genotype contained the FJzz1 strain and other variant pandemic strains (Fig. 2C). All the aa changes in the FJzz1 variants at different passages are listed in Table 2.
ORFs Encoded proteins F20 F50 F100 F150 F200 ORF1a Nsp2 0 (0)* 0 (0) 1 (0.13) 1 (0.13) 1 (0.13) Nsp3 0 (0) 1 (0.06) 1 (0.06) 1 (0.06) 1 (0.06) ORF1b Nsp13 0 (0) 0 (0) 0 (0) 1 (0.17) 1 (0.17) S (1386) S 0 (0) 6 (0.43) 12 (0.87) 13 (0.94) 14 (1.01) ORF3 (225) ORF3 0 (0) 0 (0) 1 (0.44) 1 (0.44) 1 (0.44) M (227) M 0 (0) 0 (0) 1 (0.44) 1 (0.44) 1 (0.44) Total number 0 7 14 18 19 *Number of aa changes and change rates of the corresponding proteins (%) are indicated in parentheses.
Table 1. Statistics of aa change numbers and change rates in the corresponding proteins at different passages.
Figure 2. Phylogenetic analysis of FJzz1 strain. A Rate of mutation was calculated as the percentage of changed aa in the corresponding protein, including Nsp2, Nsp3, Nsp13, ORF3, S, and M. B Positions of the changed aa in the S protein are shown. The y-axis represents the positions of the changed aa, and the x-axis represents the six different pairs of virulent parental /attenuated strains (Pair 1: DR13/attenuated DR13; Pair 2: PC22A/PC22A-P160; Pair 3: 83P-5/83P-5-100th; Pair 4: KNU-141112-P5/KNU-141112-S-DEL2-ORF3; Pair 5: YN15/Y144; Pair 6: FJzz1-F20/FJzz1-F200). C A phylogenetic tree was constructed based on S gene of FJzz1 strain and another 60 reference strains with complete S gene sequences available in GenBank. The PEDV strain FJzz1 was marked with the filled triangle.
ORFs Amino acid position F20 F50 F100 F150 F200 ORF1a Nsp2 (750) 699 M M L L L Nsp3 (1656) 1562 M I I I I ORF1b Nsp13 (597) 968 A A A T T S (1386) 42 Q Q H Q Q 55–57 IGE K– K– K– K– 128 F F Y Y Y 265 D D D A A 378 D D N N N 490 T T R R R 773 K K K K N 877–878 SG RR RR RR RR 900 L L L L V 1009 N N D D D 1338 I I T T T 1353 C F F F F ORF3 (225) 170 Y Y H H H M (227) 159 G G D D D
Table 2. Amino acid changes that occurred at different passages.
The S glycoprotein is one of the essential structural proteins of CoVs, and plays a vital role in genetic variation, viral invasion, viral virulence, and the production of neutralizing antibodies. To explore the antigenic variation in the FJzz1 variants during their serial passage, a sequence alignment of the deduced S glycoprotein aa in different generations was constructed. This showed that compared with the classic strain CV777, the low-passaged FJzz1 variants (F5 and F20) contained five aa insertions. However, after serial passage in vitro, the high-passaged FJzz1 variants (F50, F100, F150 and F200) showed characteristic consecutive aa deletions in the S1-NTD region (55I56G57E → 55K56△57△), and all the FJzz1 variants in different generations had two aa deletions relative to CV777 (Fig. 3). Interestingly, an aa change (N-D) occurred in variants F100, F150 and F200, but not at the same position in variants F5, F20, and F50, resulting in the disappearance of a predicted N-linked glycosylation site in the S glycoprotein in variants F100, F150 and F200. A total of six consistent aa changes were observed at the epitopes in COE and SS6, but the effects of these changes on the neutralization activity of these epitopes remain to be investigated. CoVs contain two conserved motifs, KVHVQ and YxxΦ, in the cytoplasmic tail of the S protein, which may affect the pathogenicity of CoVs (Schwegmann-Wessels et al. 2004; Youn et al. 2005; Winter et al. 2008). In the present study, we noted that these two motifs were conserved and stable during the serial passage of FJzz1.
Figure 3. Alignment of S protein sequences of FJzz1 variants at different passages and other PEDV strains. S protein aa sequences of FJzz1 variants at different passages (marked with black box) and other PEDV strains including the classical CV777 strain, were aligned using Clustal W. Predicted N-linked glycosylation sites in the FJzz1 variants are marked with red arrows. Regions inserted and deleted relative to CV777 are highlighted in red and blue, respectively. The substitutions in neutralizing epitopes are highlighted in yellow.
To determine whether the virulence of the FJzz1 strain changed during its serial passage in vitro, FJzz1-F20 and FJzz1-F200 were analyzed simultaneously to evaluate their pathogenicity in suckling piglets. The results showed that piglet A2 in the group infected with FJzz1-F20 (group A) began to show diarrhea at 15 hpi, with typical clinical signs such as loose yellowish stools and loss of appetite. Most of piglets in group A developed diarrhea by 24 hpi, and all five piglets inoculated with FJzz1-F20 developed severe watery diarrhea within 48 h. However, the clinical signs of piglet A3 were alleviated after 8dpi, with an increase in appetite. By contrast, most of the piglets infected with the same dose of FJzz1-F200 were in good condition, and their activity levels and feed intake were significantly higher than those of the piglets in group A. Piglets B4 and B5 in the group infected with FJzz1-F200 (group B) showed transient mild diarrhea signs at 2 and 6 days postinfection (dpi), respectively, lasting for less than 24 h. The other piglets in group B showed no obvious signs, and none of the piglets in the control group (group C) showed any clinical signs throughout the experiment, consistent with expectations (Fig. 4A). The daily body temperature and bodyweight of each piglet were monitored, and the body temperatures of most piglets remained relatively stable, except for the moribund piglets, whose body temperatures dropped significantly (Fig. 4B). There were no significant differences in the bodyweights of the piglets among the groups before inoculation. However, the bodyweights of the piglets in group A decreased significantly within 6 dpi (Fig. 4C). By contrast, the bodyweights of most piglets in group B remained relatively stable, and increased significantly after 1 week. The weights of all the piglets in group C maintained a gradual increase throughout the experiment, as expected. Piglets A2, A5, A4, and A1 in group A gradually became moribund and died or were euthanized at 2, 4, 5, and 7 dpi, respectively. The mortality rate of group A was 80%, whereas no mortality was observed in the piglets of group B or group C (Fig. 4D). These results indicate that FJzz1-F20 was virulent in piglets, whereas FJzz1-F200 was significantly attenuated after serial passage in vitro.
Figure 4. Pathogenicity analysis of FJzz1-F20 and FJzz1-F200. A Fecal scores of piglets with the valuation standard: 0 = normal; 1 = soft; 2 = semi-fluid; 3 = watery diarrhea. B Changes in average body temperature in each group within the first 14 dpi. C Average bodyweight changes in each group. D Survival rates of piglets in each group. Survival curves of piglets infected with DMEM (Mock), FJzz1-F20, or FJzz1-F200 are shown.
Pathological and histological examinations were performed to systematically and intuitively evaluate the pathogenicity of variants FJzz1-F20 and FJzz1-F200 in suckling piglets. FJzz1-F20 infection caused severe pathological changes throughout the entire small intestine characterized by thin or even transparent intestinal walls and a large amount of yellowish fluid in the intestinal cavity. Varying degrees of swelling and bleeding were observed in the inguinal lymph nodes and mesenteric lymph nodes, which appeared dark red or dark purple, whereas other organs such as the lung, spleen, and kidney, showed no visible pathological changes (Supplementary Figure S1). By contrast, the piglets in the FJzz1-F200 infection group and the control group showed no visible pathological lesions in these organs, as expected. Histological staining revealed that FJzz1-F20 infection caused severe histopathological lesions in all the intestinal segments, especially the jejunum and ileum, characterized by the atrophy, shortening, or even shedding of the intestinal villi (Fig. 5A). The large intestine, particularly the cecum, was slightly damaged, showing varying degrees of atrophy. In the FJzz1-F200 infection group, no visible microscopic lesions were detected other than slight intestinal villus damage in the jejunum. The intestinal villi of the piglets in the control group were intact with no microscopic lesions, as expected. Staining for the PEDV N protein appeared as tiny stipples within the cytoplasm of the jejunal and ileal cells. PEDV-positive enterocytes were sporadically detected in the jejunum and ileum in both the FJzz1-F20- and FJzz1-F200-infected piglets, but the amount of PEDV detected was significantly higher in the FJzz1-F20-infected group than in the FJzz1-F200-infected group, whereas no PEDV was detected in the control group (Fig. 5B). In conclusion, these results show that the pathogenicity of FJzz1-F200 was significantly lower than that of FJzz1-F20.
Figure 5. Histopathological lesions in different intestinal segments of piglets inoculated with FJzz1-F20 or FJzz1-F200. A Different intestinal segments (Jejunum, Ileum, Cecum, and Colon) from FJzz1-F20-, FJzz1-F200- or mock-inoculated piglets were collected on the day of death or at the final time points for hematoxylin–eosin staining. B Immunohistochemical detection. Jejunal and Ileal tissues from each group were stained with a monoclonal antibody directed against PEDV N protein (1:200 dilution), scale bar = 200 μm.
Viral shedding in the feces was measured with TaqMan real-time RT-PCR (Kim et al. 2007). The viral shedding titers peaked within 1–3 dpi, in a range of 3.24 × 108 to 1.13 × 109 copies/mL, after which they decreased, ranging from 2.32 × 104 to 6.07 × 106 copies/mL at 5–13 dpi (Fig. 6A). However, the piglets in group B shed virus at a low level in the initial stage of infection, after which the titers increased gradually to reach a peak of 7.83 × 106 copies/mL at 7 dpi, significantly lower than that in the piglets of group A. The Viral shedding titers in the piglets in group B then decreased again until it did not differ significantly from that of group A (Fig. 6A). We simultaneously examined the viral loads in the different segments of the intestines, including the ileum, jejunum, cecum, and colon. As shown in Fig. 6B, all the segments of the intestine showed high viral loads in the piglets of group A, ranging from 1.11 × 106 to 6.56 × 108 copies/g, significantly higher than those in the piglets of group B (8.26 × 104 to 1.36 × 105 copies/g). During the whole experiment, both the fecal viral shedding rate and the viral loads in the different segments of intestines were deemed to be negative in group C (Fig. 6B). These results confirm that the pathogenicity of FJzz1-F200 was significantly attenuated in piglets relative to that of FJzz1-F20.
Figure 6. Fecal viral shedding and quantification of viral load in different intestinal segments. Viral shedding in feces A and the viral loads in different intestinal segments B were determined with TaqMan real-time RT-PCR targeting the PEDV N gene. Asterisk (*) indicates a significant difference between FJzz1-F20 and FJzz1-F200 (*P < 0.05; **P < 0.01; ***P < 0.001).
The production of cytokines including types I/III IFN and pro-inflammatory cytokines in the target tissue is part of the innate immune response to viral infection. To investigate the innate immune responses induced by PEDV infection in vivo, the cytokines in the jejunums of the piglets were quantified with relative RT-qPCR. The levels of the type I IFN (IFN-α) and type III IFN (IFN-λ3) transcipts induced in the jejunum tissue were significantly higher after FJzz1-F20 and FJzz1-F200 infection than in the control group, and FJzz1-F200 infection induced higher levels of IFN than FJzz1-F20, especially IFN-λ3, which was significantly elevated (Fig. 7). FJzz1-F20 infection also induced higher IL-1β and IL-8 in the transcript levels in the jejunum tissue than were observed in the control group. FJzz1-F20 infection induced higher levels of IL-8 mRNA than did FJzz1-F200 infection. Moreover, compared with the control group, FJzz1-F200 infection significantly increased the transcription levels of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-8, in the jejunum tissue. These results suggest that PEDV infection induces the innate immune response in its target tissues, inhibiting viral replication.
Figure 7. Quantification of cytokines in jejunal tissue. Production of cytokines, including A type I interferon (IFN-α) and B type III interferon (IFN-λ3), and pro-inflammatory cytokines, such as C TNF-α, D IL-1β and E IL-8, were measured with real-time RT-PCR in jejunal tissue. The experiment was performed three times and the representative data are shown. Error bars represent means ± standard deviations (SD). *P < 0.05; **P < 0.01; ***P < 0.001.