Genetic Characteristics of Porcine Hemagglutinating Encephalomyelitis Coronavirus: Identification of Naturally Occurring Mutations Between 1970 and 2015

Figures

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  Figure 1. Phylogenetic analysis of 14 PHEV strains isolated between 1970 and 2015. Phylogenetic trees were constructed using the neighbor-joining algorithm in the MEGA-X software and based on (A) Full-length genome, or individual ORFs including (B) spike glycoprotein ORF, (C) HE glycoprotein ORF, (D) membrane protein ORF, (E) envelope protein ORF, (F) nucleocapsid protein ORF, and (G) replicase polyprotein ORF. The numbers on each branch are the bootstrap values (%) in 1000 replicates. The scale bar indicates the amino acid substitution at each position. The strain PHEV/67 N/US/1970 isolated in the United States in 1970 was labeled with a black diamond.

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  Figure 2. Similarity map of the full-length genome sequence of representative PHEV isolates. (A) Schematic diagram of the complete PHEV genomic structure. From the 5′end to the 3′ end are Replicase polyprotein ORF, accessory protein NS2A ORF, HE protein ORF, spike glycoprotein ORF, accessory protein NS12.7 ORF, envelope protein ORF (E), membrane protein ORF (M) and nucleocapsid protein ORF (N). (B) SimPlot similarity analysis results using SimPlot ver.3.5.1 (Lole et al., 1999). Virus PHEV/USA-15TOSU1209 (GenBank ID: KY419107.1) was used as the query sequence to compare with five other representative strains from the United States, China, and Belgium.

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  Figure 3. Deletion patterns in NS2A ORF region. The yellow box indicates the expression of full-length or truncated NS2A. The white box indicates the region of non-functional expression. Deleted regions are indicated with dashed lines. The numbers indicate the nucleotide positions relative to the genome of PHEV/67 N/US/1970 strain (GenBank ID: MW165134.1).

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  Figure 4. Linear B cell epitope map of the full-length S glycoprotein of PHEV. (A) Diagram of the main features of PHEV S glycoprotein, including NTD (aa 16–298), S1-RBD (aa 311–608), S2 domain (aa 784–1,276), and transmembrane domain (TM; aa 1,294–1,316). Numbers refer to the amino acid positions corresponding to PHEV/67 N/US/1970 S glycoprotein. (B) Mapping of linear B cell epitopes using the BepiPred-2.0 server. The Y-axes represents the residue score, and the X-axes represents the amino acid position, which is also relative to the diagram on the top (A). The predicted epitopes are indicated in yellow.

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  Figure 5. Comparison of linear B cell epitopes affected by amino acid variations in the NTD of S glycoprotein between PHEV/USA-15TOSU25049 and PHEV/67 N/US/1970. (A) Amino acids of the predicted linear B cell epitope are indicated with bold red letters, while numbers refer to their positions relative to the corresponding full-length S glycoprotein. Three-dimensional view of epitopes of PHEV/USA-15TOSU25049 (B) and PHEV/67 N/US/1970 (C). The tertiary structure was modeled using I-TASER. The predicted epitopes are circled, and amino acid variations are marked with black dots.

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  Figure 6. Comparison of linear B cell epitopes affected by amino acid variations in the S2 domain between PHEV/USA-15TOSU25049 and PHEV/67 N/US/1970. (A) Amino acids of the predicted linear B cell epitope are indicated with bold red letters, while numbers refer to their positions relative to the full-length S glycoprotein. Three-dimensional view of epitopes in the S2 domain of PHEV/USA-15TOSU25049 (B) and PHEV/67 N/US/1970 (C). The predicted epitopes are circled, and the amino acid variations are marked with black dots.

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  Figure 7. Comparison of linear B cell epitopes affected by amino acid variations in the S1-RBD between PHEV/USA-15TOSU25049 and PHEV/67 N/US/1970. (A) Amino acids of the predicted linear B cell epitope are indicated with bold red letters, while numbers refer to their positions relative to the corresponding full-length S glycoprotein. Three-dimensional view of epitopes in the S1-RBD of PHEV/USA-15TOSU25049 (B) or PHEV/67 N/US/1970 (C). The predicted epitopes are circled, and amino acid variations are marked with black dots.