Antibodies Raised against Bcvir15, an Extrachromosomal Double-Stranded RNA-Encoded Protein from Babesia canis , Inhibit the In Vitro Growth of the Parasite

Abstract

ABSTRACT As part of a search for homologous members of the Plasmodium falciparum Pf60 multigene family in the intraerythrocytic protozoan parasite Babesia canis , we report here the characterization of a cDNA of 1,115 bp, which was designated Bcvir for its potential viral origin. The Bcvir cDNA contained two overlapping open reading frames (ORFs) (ORF1 from nucleotide [nt] 61 to 486 and ORF2 from nt 417 to 919), where Bcvir15, the deduced ORF1 peptide (M 1 to I 141 ), is the main expressed product. The Bcvir cDNA was derived from an extrachromosomal dsRNA element of 1.2 kbp that was always found associated with a double-stranded RNA (dsRNA) of 2.8 kbp by hybridization, and no copy of this cDNA sequence was found in B. canis genomic DNA. Biochemical characterization of Bcvir15, by using polyclonal rabbit sera directed against recombinant proteins, indicated that it is a soluble protein which remained associated with the cytoplasm of the B. canis merozoite. Interestingly, purified immunoglobulins from the anti-glutathione S -transferase-Bcvir15 (at a concentration of 160 μg/ml) induced 50% inhibition of the in vitro growth of B. canis , and the inhibitory effect was associated with morphological damage of the parasite. Our data suggest that the extrachromosomal dsRNA-encoded Bcvir15 protein might interfere with the intracellular growth of the parasite rather than with the process of invasion of the host cell by the merozoite. Epitope mapping of Bcvir15 identified three epitopes that might be essential for the function of the protein.