The highly pathogenic strain of porcine deltacoronavirus disrupts the intestinal barrier and causes diarrhea in newborn piglets

Figure 1.. PDCoV isolation and culture. (a) Assays for PCV2, PDCoV, TGEV, PRRSV, PRV, PEDV, BVDV were performed sequentially. (b) PDCoV P10 infected ST cells, and the MOI = 0, 0.5, 1, and 24 h later for immunofluorescence detection (n = 3). (c) PDCoV-N protein green fluorescence was quantified using the DAPI fluorescence value as a standard (n = 3, ****, p < 0.0001). (d) The supernatant of infected tenth passage lysates was collected for TEM (n = 3).

Figure 2.. Sequencing and cellular adaptation of PDCoV (a) genome sequencing depth results. (b) Sequencing fragmentation assembly results. (c) Assembly results were aligned to the database. (d) N recombinant plasmids were prepared to generate a standard curve (n = 3). (e) Virus copy numbers of virus fluid from the adsorbent fluid of the disease and from the tenth generation of culture were compared (n = 3), and virus infectivity was compared between the first and tenth generation viruses (n = 8) (** p < 0.01).

Figure 3.. The whole genome and S protein sequences of the CH/LNFX/2022 strain were analyzed for genetic evolution. The legend of A and B is the same. (a) The CH/LNFX/2022 and 50 PDCoV strains were subjected to evolutionary analysis and divided into two subtypes, GI and GII, with three lineages of strains from China/America, Japan, and Korea/Southeast asia. (b) S protein is the same as above.

Figure 4.. Two strains per region were randomly picked for alignment. (a) S protein to contrast. (b) M protein to contrast. (c) N protein to contrast. (d) NS6 protein to contrast. (e) E protein to contrast. (f) NS7 protein to contrast. The red box represents the unique evolutionary characteristics of CH/HLJJS/2022, and the blue box represents the characteristics of each lineage.

Figure 5.. Detailed alignment of the S protein model to the reference strain. (a) S protein modeling results for CH/LNFX/2022. (b) The S protein 100-150AA range was modeled and the protein tertiary structure has a specific bulge at A – V (aa 137). (c) The S protein 500-570AA range was modeled and the protein tertiary structure has a specific bulge at S-L (aa 505) and T-I (aa 559). (d) The S protein 1050-1100AA range was modeled and the protein tertiary structure has a specific bulge at D – H (aa 1076).

Figure 6.. Protein function prediction. (a) Primary epitope comparative analyses of five regionally representative strains and CH/LNFX/2022 strain revealed 3-aa mutations out finding specific changes. (b) O-GalNAc (mucin type) glycosylation sites in mammalian proteins were predicted using netnglyc-1.0. (c) Prediction of the position of the coronavirus 3C like protease cleaving protease in the protein using netcorona-1.0. (d) TMHMM (v2.0) was used to predict the transmembrane functional changes of S protein.

Figure 7.. Pathological analysis of newborn piglets infected with highly pathogenic CH/LNFX/2022. (a) Newborn piglets were orally infected with PDCoV (n = 10) after 48 h artificial milk replacement, and the mock infection group was treated with PBS instead (n = 4). (b) Watery diarrhea occurred 24 h after infection and was normal in the mock infection group. (c) Colonic staining results were quantified using the mock infection group as a control (n = 3, *, p < 0.05; **, p < 0.01; ***, p < 0.001). (d) The animal model of infection was established by immunofluorescence of the jejunum (n = 3). (e) Results of scanning electron microscopy of the colon (n = 3).