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 (EIAV(PV)) of the cell culture-adapted strain of EIAV (EIAV(PR)). 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 of rev), and the 3' long terminal 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 signs of acute equine infectious anemia (EIA) at 23 days postinfection (dpi). 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 dpi, 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. Analysis of the complete nucleotide sequence demonstrated that the 3.3-kbp 3' fragment of EIAV(UK) differed from the consensus sequence of EIAV(PV) by just a single amino acid residue in the second exon of the rev gene. Complete homology with the EIAV(PV) consensus sequence was observed in the hypervariable region of the LTR. However, EIAV(UK) 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 EIAV(PV) 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, EIAV(UK), 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.

FIG. 1

Amplification and molecular cloning of the 3.3-kbp 3′-terminal fragment of EIAV_PV. A 3.3-kbp fragment from EIAV_PV-infected cell cultures was amplified by PCR, blunt ended, and digested with NcoI prior to ligation with NcoI/SmaI-digested pLG338/30 vector DNA. The resultant pLG338/PV3.3 molecular clones were digested with NcoI and EcoRI, and the 3.3-kbp EIAV_PV fragment was inserted into the infectious molecular clone 19-2-6A. This produced chimeric proviral molecular clones designated EIAV_PV3.3.

FIG. 2

Clinical responses in animals infected with EIAV derived from infectious molecular clones. Body temperature and platelet count are shown for animals infected with viruses from molecular clone 19-2-6A (A), viruses from a mixture of EIAV_PV3.3 molecular clones (B), and viruses from molecular clone EIAV_UK (C to F).

FIG. 3

Comparison of the deduced amino acid sequences encoded by EIAV_UK with the 19-2 sequence, the EIAV_PV consensus sequence, and the sequence of a cloned representative of the EIAV_PR strain. Comparisons shown are for ORF S2 (A), ORF S3, which encodes the second exon of rev (B), and the envelope glycoprotein (C). Only the amino acid residues that differ from the total consensus sequence are indicated. A dash indicates that no consensus sequence was found. Potential N-linked glycosylation sites are underlined. Potential N-linked glycosylation sites that differ between (EIAV_UK and EIAV_PV) and EIAV_PR and 19-2) are marked by asterisks.

FIG. 4

Comparison of the U3-LTR hypervariable regions of the EIAV_WY strain with the EIAV_PV consensus sequence EIAV_UK,the 19-2-6A sequence, and a consensus sequence for the EIAV_PR strain. Potential cis-acting sequences (PEA-2, CAAT, ets, AP-1, and TATA) are underlined. The presence of a possible new cis-acting element is indicated by ?. Gaps (indicated by dashes) were introduced to align the sequence. In EIAV_PR, R indicates either G or A, as at this location 54% of the population is represented by G and 46% is represented by A (29).

FIG. 5

Comparison of nucleotide sequences between EIAV_UK and EIAV_PV consensus sequences showing the location of the insertion/duplication in the 3′ LTR. The first repeat of the 68-nucleotide region in U3 is indicated by a white box; the second repeat of the same sequence in EIAV_UK is marked by a shaded box.