PMC:7019868 / 17447-23718 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7019868","sourcedb":"PMC","sourceid":"7019868","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7019868","text":"3. Results\n\n3.1. Recovery of recombinant PEDVs\nWe have recently developed a reverse genetic platform of PEDV and have successfully generated a recombinant virus, iPEDVPT-P96, which is phenotypically comparable to its parental attenuated Taiwan PEDV-Pintung 52 strain, PEDVPT-P96 [19]. To study the role of the S gene in the attenuation mechanism of PEDVPT-P96, we further generated the recombinant, highly virulent Taiwan PEDVPT 52 strain, iPEDVPT-P5, and two chimeric viruses (iPEDVPT-P5-96S and iPEDVPT-P96-5S) by replacing the complete sequence of the S gene reciprocally (Figure S1). In general, the approach to rescue the new recombinant viruses were virtually the same as published previously [19]. However, for the generation of iPEDVPT-P5 and iPEDVPT-P5-96S, we split the plasmid B to two fragments at nucleotide position of 9654 according to Hou et al. [23] in order to compensate for the instability caused by the toxic sequences. The in vitro and in vivo properties of iPEDVPT-P5 were confirmed similarly to those of the parental PEDVPT-P5 strain in a seven-day-old conventional piglet model.\nAt 24–48 h post-electroporation, typical PEDV cytopathic effects of giant syncytia were observed for all recombinant viruses. Viral stocks were prepared by passaging viruses in Vero cells one additional time (P1), and the presence of PEDV was confirmed by detection of PEDV S protein by immunofluorescence assay (Figure 1A) and sequence analyses.\n\n3.2. In Vitro Characterization of Recombinant PEDVs\nThe in vitro characteristics of the recombinant viruses were compared by evaluation of the size of syncytia, growth kinetics in Vero cells, and plaque morphologies. All recombinant viruses induced formation of syncytia by 18 h post-infection. Viruses carrying the identical S gene exhibited similar fusogenic ability as suggested by the comparable number of nuclei per syncytium; representative micrographs used to count the number of nuclei in syncytia are shown in Figure 1A. Remarkably, viruses carrying S gene derived from iPEDVPT-P96, induced 3.5 to 4 times larger syncytia than the other two viruses containing S gene derived from iPEDVPT-P5 (Figure 1B).\nReplication of all recombinant viruses in Vero cells were examined by performing both one-step and multistep growth kinetics at a multiplicity of infection (MOI) of 1 and 0.001, respectively. Interestingly, although replacement of the S gene resulted in an alteration of the replication kinetics and efficiency, leading to similar growth curve patterns between viruses carrying the identical S gene sequence, it did not fully reverse the capability of virus yield, at least in Vero cells (Figure 1C).\nWe observed that plaque morphologies of the four recombinant viruses correlated to the results of syncytia size. At 72 h post-infection, both iPEDVPT-P5 and iPEDVPT-P96-5S induced barely visible viral plaques in Vero cells macroscopically, whereas iPEDVPT-P96 and iPEDVPT-5-96S generated distinct and comparable viral plaques with the plaque size of iPEDVPT-P5-96S being slightly smaller than that of the iPEDVPT-P96 (Figure 1D). These data suggested that the exchange of the S gene between iPEDVPT-P5 and iPEDVPT-P96 affected the fusogenicity and, to a lesser extent, replication kinetics in Vero cells.\n\n3.3. Investigation of the Role of Spike Gene on the Pathogenicity of PEDVPT 52 strain\nTo evaluate the pathogenicity related to S gene replacement in PEDVPT 52 strain, we orally inoculated 2 mL of 5 × 102 TCID50/mL of iPEDVPT-P5, iPEDVPT-P5-96S, iPEDVPT-P96, iPEDVPT-P96-5S viruses and with PI medium in seven-day-old crossbred piglets assigned in the corresponding groups. Parameters used to assess the pathogenicity of different recombinant viruses were summarized in Table 2 and shown in Figure 2. The target sequences of the recombinant viruses at the time point of peak fecal viral shedding in each pig was confirmed identical to the original inoculum. No viral RNA shedding, diarrhea or mortality was detected in the mock-treated group during the entire experimental course. Piglets inoculated with recombinant viruses carrying the S gene derived from the highly virulent iPEDVPT-P5, namely the iPEDVPT-P5 itself and iPEDVPT-P96-5S, had an early onset of clinical symptoms including diarrhea, anorexia and decreased activity, and peak viral shedding at 1 d post-inoculation (DPI). At 3 DPI, piglets inoculated with iPEDVPT-P5 (n = 3) and iPEDVPT-P96-5S (n = 3) both exhibited extensive PEDV-induced villous blunting and atrophy in the jejunum and, to a lesser extent, in the ileum; histological changes in the duodenum were not obvious (Figure 3). Mortality rates of both groups were comparable, reaching 50% at 2 (iPEDVPT-P5 group) and 5 (iPEDVPT-P96-5S group) DPI and ultimately exceeded 80% by 10 DPI.\nIn comparison, inoculation with iPEDVPT-P5-96S and iPEDVPT-P96 viruses containing the S gene derived from the attenuated iPEDVPT-P96, induced a delayed onset of clinical symptoms and peak viral shedding. Histological evaluation of piglets inoculated with both viruses (iPEDVPT-P96, n = 3; iPEDVPT-P5-96S, n = 3) at 3 DPI revealed a much milder degree of villous atrophy in the jejunum with conspicuous villous hyperplasia compared to the other two groups. Notably, iPEDVPT-P5-96S-treated piglets exhibited statistically significant severer villous atrophy in jejunum than iPEDVPT-P96-treated piglets (Figure 3). Despite the similar pattern and severity of viral shedding and clinical symptoms, inoculation with iPEDVPT-P5-96S eventually resulted in a higher mortality rate (40% in iPEDVPT-P96 and 80 % and iPEDVPT-P5-96S) and lower average daily weight gain by 10 DPI than those upon inoculation with iPEDVPT-P96. These data suggested that iPEDVPT-P96 fully regained virulence by replacement of the S gene from the virulent PEDVPT 52 strains and the complementary approach can only partially reduce the virulence of the iPEDVPT-P5.\nIn agreement with the previous study, immunohistochemistry using anti-PEDV nucleocapsid monoclonal antibody demonstrated positive signals predominantly in the jejunal and ileal enterocytes at the top of villi (Figure 4) but occasionally within the mesenteric lymph nodes. No difference in the viral distribution was identified among the recombinant 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