DEVELOPMENT AND CHARACTERIZATION OF AN IN-VIVO PATHOGENIC MOLECULAR CLONE OF EQUINE INFECTIOUS-ANEMIA VIRUS

An infectious nonpathogenic molecular clone (19-2-6A) of equine infect ious anemia virus (EIAV) was modified by substitution of a 3.3-kbp fra gment amplified by PCR techniques from a pathogenic variant (EIAV(PV)) of the cell culture-adapted strain of EIAV (EIAV(PR)). This substitut ion consisted of coding sequences for 77 amino acids at the carboxyl t erminus of the integrase, the S1 (encoding the second exon of tat), S2 , and S3 (encoding the second exon of rev) open reading frames, the co mplete env gene (including the first exon of rev), and the 3' long ter minal repeat (LTR). Modified 19-2-6A molecular clones were designated EIAV(PV3.3), and infection of a single pony (678) with viruses derived from a mixture of five of these molecular clones induced clinical sig ns of acute equine infectious anemia (EIA) at 23 days postinfection (d pi). As a consequence of this initial study, a single molecular clone, EIAV(PV3.3#3) (redesignated EIAV(UK)), was selected for further study and inoculated into two ponies (613 and 614) and two horses (700 and 764). Pony 614 and the two horses developed febrile responses by 12 dp i, which was accompanied by a 48 to 64% reduction in platelet number, whereas pony 613 did not develop fever (40.6 degrees C) until 76 dpi. EIAV could be isolated from the plasma of these animals by 5 to 7 dpi, and all became seropositive for antibodies to this virus by 21 dpi. A nalysis of the complete nucleotide sequence demonstrated that the 3.3- kbp 3' fragment of EIAV(UK) differed from the consensus sequence of EI AV(PV) by just a single amino acid residue in the second exon of the r ev gene. Complete homology with the EIAV(PV) consensus sequence was ob served in the hypervariable region of the LTR. However, EIAV(UK) was f ound to contain an unusual 68-bp nucleotide insertion/duplication in a normally conserved region of the LTR sequence. These results demonstr ate that substitution of a 3.3-kbp fragment from the EIAV(PV) strain i nto the infectious nonpathogenic molecular clone 19-2-6A leads to the production of progeny virus particles with the ability to induce clini cal signs of EIA. Therefore, EIAV(UK), which is the first pathogenic, cell culture-adapted molecular clone of EIAV to be described, should b e of value in identifying viral determinants of pathogenicity.