Genetic characterization of atypical porcine pestivirus from neonatal piglets with congenital tremor in Hubei province, China

Fig. 1. Identification of APPV in CT-affected piglets. A Detection of APPV in CT-positive samples. Four cerebellums were chosen to detect the APPV genome based on partial NS3 gene (Lane 1–4). ddH2O was used as a negative control (Lane 5). B Detection of the viral protein expression in PK-15 cells. The viral protein was detected in PK-15 cells inoculated with APPV-positive serum or APPV E2 mAb, mock-infected cells (PK-15) were used as the negative control. C Histological lesions in a CT-affected piglet. D Immunohistochemical detection in a CT-affected piglet

Fig. 2. Viral load of APPV in CT-affected piglets. A Viral load of APPV in the central nervous system of CT-affected piglets. B Viral load of APPV in various tissues of CT-affected piglets. Various tissues (heart, liver, spleen, lung, kidney, cerebrum, cerebellum, brainstem, spinal cord, submaxillary lymph nodes, tonsil, and serum) from CT-affected piglets were tested to determine the copy number of APPV by absolute qRT-PCR. Data are represented as the mean ± SD. LOD, limit of detection

Fig. 3. Phylogenetic analysis of all available APPV strains. Phylogenetic analysis based on encoding sequences of the whole genome (A), E2 (B), Erns (C), Npro (D), NS3 (E), and NS5B genes (F) were conducted in MEGA11 using the Maximum likelihood method and GTR + G + I (for full-length CDS, Npro, NS3, and NS5B) or TN93 + G + I (for E2 and Erns) substitution model. Bootstrap values were calculated for 1000 replicates, only values > 50% are indicated. A rat pestivirus isolate (NC_025677) from USA was used as an outgroup. ▲ indicates the sequence from the virus described in this study (color figure online)

Fig. 4. Nucleotide (A) and amino acid sequence (B) identities of the Npro, Erns, E2, NS3, and NS5B genes between CH-HB2020/2021 and other APPV isolates. Nucleotide and amino acid sequences were aligned using Lasergene Megalign 7.1.0 software, and then visualized with heat map in GraphPad Prism 8.0 software

Fig. 5. Recombination analysis of all available APPV strains based on the encoding sequences. Potential recombination events were identified using Recombination Detection Program version 4 (RDP4) and then examined using SimPlot version 3.5.1 and Bootscan analysis. Additional details were represented in Additional file 5: Table S3

Fig. 6. Geographical distribution of APPV in China. APPV prevalent regions were shown in pink. The transregional transmission route of APPV proposed in this study was also shown