Table 1.
Primers used for differentiation of the potential agents.
Table 2.
Primers used for amplifying the complete genome sequence for HY12.
Figure 1.
Cytopathic effect of HY12 in cell culture and EM observation.
HY12 caused a typical cytopathic effect in MDBK cells after 6–8 h inoculation. Cells became rounded with an increased refraction (B). 24∼48 h post infection, majority of the infected cells detached off the flask (C). The normal MDBK cells were used as negative controls (A). HY12 virus particles observed by electron microscopy to be about 22∼28 nm in diameter as indicated by arrow (D), the scale bar is 100 nm.
Table 3.
Partial physiochemical properties for HY12 strain.
Figure 2.
Unique amino acid mutations in the capsid proteins encoded by HY12 isolates.
The amino acid sequence of HY12 isolates were deduced from the nucleotide sequence, and was aligned with 12 known BEV strains in the GenBank. Alignment analysis was performed using each HY12-encoded structural protein as a template. Results were shown respectively for VP1 (A), VP2 (B), VP3 (C), and VP4 (D). The identical amino acids were marked with symbol “·”, and different amino acids to HY12 were presented as individual amino acid symbol. The unique mutation for HY12 was highlighted with red color. Deletion of amino acids were marked as “-”.
Figure 3.
Phylogenetic analysis clustered HY12 strain to a new serotype/genotype within enterovirus E.
Phylogenetic tree were generated by neighbor-joining methods by comparing the sequence regions of 5′-UTR, VP1, VP2, VP3, VP4, 3D, and the complete genome sequences for 15 enteroviruses. HY12 strain was placed to the cluster of enteroviruses E after phylogenetic analysis with the all nucleotide sequence regions except 5′-UTR (B–F). The HY12 strain was revealed as a new serotype/genotype (serotype/genotype 3) that only consists of D14/3/96 and HY12 strains in relation to serotype 1 (LC-R4, VG5-27,Vir 404/03) and serotype 2 (SL305, K2577,PS 42, PS 83) enterovirus strains (B–E). When nucleotide sequences for the non-structural proteins 3D and the complete genome sequence were employed, the HY12 were clustered to the same clade most close to SL305 and K2577 within enteroviruses E (F). However, HY12 strain was clustered to neither clade in enteroviruses E nor enterovirus F using the 5′-UTR sequence (A), suggesting an intraserotypic recombination during HY12 evolution. The position of HY12 was highlighted with a triangle.
Figure 4.
Recombination revealed in the HY12 strain.
Neighbor-joining trees of the structural proteins VP1-VP4, and the non-structural protein 3D of 15 enteroviruses were compared. When amino acid sequences for VP1, VP3, and VP4 were used to generate phylogenetic tree, similar patterns were observed as those in Fig 3B, 3D, and 3E, indicating a interserotype recombination for the HY12 strain. Like the observation in Fig 3A, F and G, the HY12 was clustered closely to K2577, SL305, PS 42 and PS 83 strains, an indication of complex interserotypic and intraserotypic recombination in the evolution for HY12. The positions of HY12 were highlighted with a triangle.