Figure Legends
Figure 1. The detection results of PCV4. The detection rates (positive/ total samples) were shown according to sampling year and geographic locations (A, B). Provinces having PCV4 positive farms are indicated with red color. The positive rates were also summarized by age groups (C) which the samples were sorted into, including aborted fetus, suckling (≤ 30 days), weaned (30 to 60 days), grower (60 to 90 days), finisher (≥ 90 days) and sow.
Figure 2. Genomic characterization of E115 strain. It was illustrated that E115 has a circular genome, containing 6 putative overlapping open reading frames (ORFs) oriented in different directions (A). The stem- loop structure with conserved nonanucleotide (highlighted) was observed (B). Comparison of the nonanucleotide sequences, (T/n)A(G/t)TATTAC, between PCV4 and the viruses of the genusCircovirus (C). The scissors represented the nicking position in the nonanucleotide motif.
Figure 3. Functional domains of putative replication- associated protein (Rep). E115 strain (arrow) was predicted containing two domains of endonuclease and helicase which were well conserved for different viruses of Circovirus genus (highlighted). In each motif, the capital letters indicated conserved amino acid, ‘x’ was any amino acid and square brackets indicate the list of acceptable amino acids in the given position.
Figure 4. Prediction of nuclear localization signal (NLS) at the N-terminus of the putative Cap. This arginine-rich region was shown based on the alignment with other members of Circovirus genus. Sequence analysis revealed that the arginine-rich region of E115 strain was aligned with the basic motifs of the NLS of PCV1, PCV2 and PCV3 which were experimentally confirmed (highlights).
Figure 5. The P-x-x-P and Y-x-x-ϕ motifs located in the putative capsid proteins. Capsid proteins of PCV4 were detected containing several P-x-x-P and Y-x-x-ϕ motifs. Y-x-x-ϕ motifs and the first P-x-x-P motif were observed in other viruses belonging to genusCircovirus . On the other hand, the second P-x-x-P only detected in PCV4 which were highlighted in a box. In each motif “x” denoted as any amino acid, while ϕ represented any of F (phenylalanine), I (isoleucine), L (leucine) or V (valine). The brackets show the conserved amino acid in given position. The number indicated the location of each motif based on the capsid protein of E115 strain.
Figure 6. Prediction of conserved regions of the putative ORF3 protein. Based on the alignment with some members of genus Circovirus, four conserved regions in the putative ORF3 protein of E115 strain (indicated as arrow) were observed. All putative ORF3s of PCV4 were shown as in dash box. The well conserved amino acids were also highlighted. In each short linear motif, the capital letters indicated conserved amino acid, ‘x’ was any amino acid and square brackets indicate the list of acceptable amino acids in the given positions. C-terminal regions of the putative PCV4 ORF3 protein were predicted as containing PDZ binding peptides. The number showed the position corresponding in E115 strain.
Figure 7. Phylogenetic tree of circoviruses based on amino acid sequence of Rep. Inserted panel was the phylogeny inferred from a data containing seven assigned families of CRESS DNA viruses. It was observed that the newly identified PCV4 (arrows) was grouped within familyCircoviridae . Focused on that family, two well supported genus ofCyclovirus and Circovirus were observed. Of which, PCV4 was within genus Circovirus , distantly related to previous known PCV1, PCV2 and PCV3 (indicated by filled circles). Numbers on branches were bootstrap support values. Bar showed number of amino acid substitutions per site.