Elongation of the C terminus by two amino

acids did not change the reactivity of mAb 8E4 against PCV2a/CL in the IPMA (Figure 1a). Furthermore, rJF2-ORF2, derived from PCV2a/JF2, in which the C terminus was elongated by three amino acids, had the same reactivity with mAb 8E4 as rCL-ORF2 and rCL-YJ-5 in the IPMA (Figure 1c). In previous studies, selleck screening library analysis of the reactivity of PCV1/PCV2 chimeras has suggested that the amino acid sequences from aa 47-62 and 165-200, as well as the last four C-terminal amino acids of Protein Tyrosine Kinase inhibitor the capsid protein, are likely to be in close proximity and may form a cluster of conformational epitopes on the surface of the PCV2 virion [6]. In the present study, the replacement of an amino acid residue (A59R) in the capsid

protein altered the reaction of PCV2a (LG, CL, and JF2) with mAb 8E4. Therefore, it could be concluded that the alanine at position 59 was a critical amino acid in the conformational neutralizing epitope recognized by mAb Captisol cell line 8E4. Alanine is a nonpolar hydrophobic amino acid with a molecular weight (MW) of 89 Da, whereas arginine is a polar basic hydrophilic amino acid with a MW of 174. Due to the differences in size, charge and hydrophobicity between alanine and arginine, this may have major consequences on the secondary and tertiary structure of the PCV2 capsid protein. Therefore, it could be concluded that the replacement of an amino acid residue (A59R) in the capsid protein of PCV2a (CL, Amisulpride LG and JF2) disrupted the binding of mAb 8E4 completely. Furthermore, the amino acid at position 59 is located on loop BC of the capsid protein [31]. This loop together with loop DE and HI are on the exterior surface of the PCV2 to form

the highest protrusion [31]. Therefore, this position may be more easily recognized by B cell receptor and with a high possibility to become a conformational B cell epitope. It was confirmed that another mutant (rYJ-CL-1-59), which contained a single amino acid mutation of R to A at position 59, did not have the ability to react with mAb 8E4. We suggest that the amino acid at position 59 of capsid protein is a necessary but not sufficient residue for epitope recognition by mAb 8E4. The 3D structure of capsid protein and mAb 8E4 complex should be studied to gain full knowledge of the conformational epitope against mAb 8E4. Conclusions In summary, a mAb (8E4) with neutralizing activity could be used to differentiate PCV2a strains (CL, LG, and JF2) from other PCV2b strains (YJ, SH and JF). These results confirm that there are antigenic differences among PCV2 strains [14]. Furthermore, reverse genetics were used to explore the genetic basis of the different reactions of PCV2a/CL and PCV2b/YJ with mAb 8E4. Evidence is presented that the amino acid at position 59 of PCV2a (CL, LG, and JF2) capsid proteins is a critical amino acid in the conformational neutralizing epitope recognized by mAb 8E4.