Genomic quasispecies associated with the initiation of infection and disease in ponies experimentally infected with equine infectious anemia virus

Leroux

et al. 1998

J Virol

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.

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

See 3 more Smart Citations

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

Leroux

et al. 1998

J Virol

Replication Ability in Vitro and in Vivo of Equine Infectious Anemia Virus Avirulent Japanese Strain

et al. 2000

An attenuated equine infectious anemia virus (EIAV), V26, was previously prepared by 50 passages of the Japanese virulent strain V70 in primary horse macrophage culture. The horses inoculated with this V26 virus were shown to raise neutralizing antibodies against V70 without any viremia. Here, we investigated the in vitro and in vivo replication ability of V26. Comparison of the long-terminal repeat (LTR) sequences between V26 and V70 revealed a large insertion within the LTR U3 hypervariable region of V26. V26 with the mutation in the LTR showed much higher promoter activity in vitro than V70. This is consistent with the much higher replication rate of V26 in horse primary macrophage cultures compared with V70. In sharp contrast, we failed to identify the V26-specific LTR sequence by PCR, at least in sequential samples of plasma or peripheral blood mononuclear cells derived from three horses until day 62 after V26 inoculation. In contrast, antibody responses to EIAV were observed in all horses. The results suggest that the replication ability of V26 in vivo is extremely low. When one of the horses was subsequently challenged with cell-associated V70, it was found that the horse became PCR positive for EIAV. There was no LTR mutation in EIAV genome in samples periodically prepared from the V70-challenged horse. Thus it was suggested that the LTR mutation in EIAV, which occurs during serial passage in vitro, affects EIAV replication in vitro and in vivo.

Cell Specificity of the Transcription-Factor Repertoire Used by a Lentivirus: Motifs Important for Expression of Equine Infectious Anemia Virus in Nonmonocytic Cells

et al. 2000

The equine infectious anemia virus (EIAV) long-terminal repeat (LTR) has been identified as highly variable, both in infected horses and in cell culture. This nucleotide hypervariation is localized to the LTR enhancer region. The EIAV LTR has been implicated in controlling both the cell tropism and virulence of the virus and it is postulated that the enhancer-region hypervariation may be responsible for the LTR effects. Our previous studies have demonstrated that the presence of DNA motifs bound by the ets transcription-factor family member PU.1 are critically important for EIAV expression in equine macrophages. Here we identify and characterize the EIAV LTR enhancer motifs PEA-2, Lvb, Oct, and CRE, that bind to fibroblast nuclear extracts. Three of these four motifs, PEA-2, Oct, and CRE, were determined to be important for expression of the LTR in a fibroblast cell line that supports productive infection of EIAV. These motifs that are important for expression of the LTR in fibroblasts were found to be interdigitated between the PU.1 sites. We hypothesize that the combination of motif interdigitation and cell-specific usage of these motifs may be responsible for the observed EIAV LTR enhancer-region hypervariation.