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Development and Characterization of an In Vivo Pathogenic Molecular Clone of Equine Infectious Anemia Virus

ABSTRACT An infectious nonpathogenic molecular clone (19-2-6A) of equine infectious anemia virus (EIAV) was modified by substitution of a 3.3-kbp fragment amplified by PCR techniques from a pathogenic variant (EIAVPV) of the cell culture-adapted strain of EIAV (EIAVPR). This substitution consisted of coding sequences for 77 amino acids at the carboxyl terminus of the integrase, the S1 (encoding the second exon of tat), S2, and S3 (encoding the second exon of rev) open reading frames, the complete env gene (including the first exon ofrev), and the 3′ long terminal repeat (LTR). Modified 19-2-6A molecular clones were designated EIAVPV3.3, and infection of a single pony (678) with viruses derived from a mixture of five of these molecular clones induced clinical signs of acute equine infectious anemia (EIA) at 23 days postinfection (dpi). As a consequence of this initial study, a single molecular clone, EIAVPV3.3#3 (redesignated EIAVUK), 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 dpi, which was accompanied by a 48 to 64% reduction in platelet number, whereas pony 613 did not develop fever (40.6°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. Analysis of the complete nucleotide sequence demonstrated that the 3.3-kbp 3′ fragment of EIAVUKdiffered from the consensus sequence of EIAVPV by just a single amino acid residue in the second exon of the rev gene. Complete homology with the EIAVPV consensus sequence was observed in the hypervariable region of the LTR. However, EIAVUK was found to contain an unusual 68-bp nucleotide insertion/duplication in a normally conserved region of the LTR sequence. These results demonstrate that substitution of a 3.3-kbp fragment from the EIAVPV strain into the infectious nonpathogenic molecular clone 19-2-6A leads to the production of progeny virus particles with the ability to induce clinical signs of EIA. Therefore, EIAVUK, which is the first pathogenic, cell culture-adapted molecular clone of EIAV to be described, should be of value in identifying viral determinants of pathogenicity.