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Journal of virology1998; 72(2); 1383-1393; doi: 10.1128/JVI.72.2.1383-1393.1998

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.
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Publication Date: 1998-01-28 PubMed ID: 9445039PubMed Central: PMC124617DOI: 10.1128/JVI.72.2.1383-1393.1998Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article discusses the creation and characterization of a harmful molecular clone of equine infectious anemia virus (EIAV). The clone was developed from a non-harmful one and induced definite clinical signs of the disease in tested animals, offering a potential tool for identifying factors contributing to the virus’s pathogenicity.

Research Objectives and Context

  • The primary aim of this study was to understand the pathogenesis of equine infectious anemia virus (EIAV). To achieve this, the researchers constructed a pathogenic, cell culture-adapted molecular clone of EIAV based on an infectious non-pathogenic clone.
  • This research is significant because it could lead to an improved understanding of viral determinants of pathogenicity, which could inform the design of innovative therapies and prevention strategies for diseases caused by EIAV.

Development of the Pathogenic Molecular Clone

  • Researchers began their study using a non-pathogenic molecular clone of EIAV, known as 19-2-6A.
  • The clone was modified by substituting a fragment, about 3.3 kilobases in length, derived from a pathogenic version of EIAV. This fragment integrated into the non-pathogenic clone, effectively transforming it into a pathogenic version. The modified clones were apportioned the name EIAV(PV3.3).

Experimentation and Results

  • Mixed viruses generated from five of the modified clones were tested on a single pony, which showed clinical symptoms of equine infectious anemia 23 days after infection.
  • Subsequently, a single clone drawn from the modified batch, then named EIAV(UK), was selected for further investigation. This clone was inoculated into two ponies and two horses. Within 12 days, a decline in platelet count and increased fever was observed in three of the four subjects.
  • The researchers were able to isolate EIAV from the plasma of these animals by 5 to 7 days post-infection. By 21 days, all had developed antibodies to the virus.

Molecular Characterization of the Pathogenic Clone

  • Nucleotide sequence analysis showed the EIAV(UK) clone differed from the pathogenic EIAV(PV) strain by just one amino acid in the rev gene’s second exon.
  • In addition, EIAV(UK) was observed to bear an unusual 68-base pair nucleotide insertion/duplication in a typically conserved region of the long terminal repeat (LTR) sequence, suggesting a possible mutation.

Implications and Conclusion

  • The study provided proof that introducing a 3.3-kbp fragment from a pathogenic EIAV strain into a non-pathogenic molecular clone leads to the replication of virulent virus particles. The resulting strain, EIAV(UK), translated into tangible clinical signs of equine infectious anemia in tested subjects.
  • EIAV(UK), therefore, is the first description of a pathogenic molecular clone of EIAV that is adaptable to cell culture. Its use could help identify determinants of the pathogenicity of the virus.

Cite This Article

APA
Cook RF, Leroux C, Cook SJ, Berger SL, Lichtenstein DL, Ghabrial NN, Montelaro RC, Issel CJ. (1998). Development and characterization of an in vivo pathogenic molecular clone of equine infectious anemia virus. J Virol, 72(2), 1383-1393. https://doi.org/10.1128/JVI.72.2.1383-1393.1998

Publication

Journal of virology
ISSN: 0022-538X
NlmUniqueID: 0113724
Country: United States
Language: English
Volume: 72
Issue: 2
Pages: 1383-1393

Researcher Affiliations

Cook, R F
  • Department of Veterinary Science, Gluck Equine Research Center, University of Kentucky, Lexington 40546, USA. rfcook1@pop.uky.edu
Leroux, C
    Cook, S J
      Berger, S L
        Lichtenstein, D L
          Ghabrial, N N
            Montelaro, R C
              Issel, C J

                MeSH Terms

                • Amino Acid Sequence
                • Amino Acid Substitution
                • Animals
                • Base Sequence
                • Equine Infectious Anemia / virology
                • Infectious Anemia Virus, Equine / genetics
                • Infectious Anemia Virus, Equine / pathogenicity
                • Integrases / genetics
                • Molecular Sequence Data
                • Sequence Alignment
                • Virulence / genetics

                Grant Funding

                • 2 P41 RR06009 / NCRR NIH HHS
                • R01CA49296 / NCI NIH HHS

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                12. Craigo JK, Durkin S, Sturgeon TJ, Tagmyer T, Cook SJ, Issel CJ, Montelaro RC. Immune suppression of challenged vaccinates as a rigorous assessment of sterile protection by lentiviral vaccines.. Vaccine 2007 Jan 15;25(5):834-45.
                  doi: 10.1016/j.vaccine.2006.09.040pubmed: 17023099google scholar: lookup
                13. Jin S, Zhang B, Weisz OA, Montelaro RC. Receptor-mediated entry by equine infectious anemia virus utilizes a pH-dependent endocytic pathway.. J Virol 2005 Dec;79(23):14489-97.
                  doi: 10.1128/JVI.79.23.14489-14497.2005pubmed: 16282448google scholar: lookup
                14. Jin S, Chen C, Montelaro RC. Equine infectious anemia virus Gag p9 function in early steps of virus infection and provirus production.. J Virol 2005 Jul;79(14):8793-801.
                  doi: 10.1128/JVI.79.14.8793-8801.2005pubmed: 15994773google scholar: lookup
                15. Zhang B, Jin S, Jin J, Li F, Montelaro RC. A tumor necrosis factor receptor family protein serves as a cellular receptor for the macrophage-tropic equine lentivirus.. Proc Natl Acad Sci U S A 2005 Jul 12;102(28):9918-23.
                  doi: 10.1073/pnas.0501560102pubmed: 15985554google scholar: lookup
                16. Maury W, Thompson RJ, Jones Q, Bradley S, Denke T, Baccam P, Smazik M, Oaks JL. Evolution of the equine infectious anemia virus long terminal repeat during the alteration of cell tropism.. J Virol 2005 May;79(9):5653-64.
                  doi: 10.1128/JVI.79.9.5653-5664.2005pubmed: 15827180google scholar: lookup
                17. Craigo JK, Li F, Steckbeck JD, Durkin S, Howe L, Cook SJ, Issel C, Montelaro RC. Discerning an effective balance between equine infectious anemia virus attenuation and vaccine efficacy.. J Virol 2005 Mar;79(5):2666-77.
                  doi: 10.1128/JVI.79.5.2666-2677.2005pubmed: 15708986google scholar: lookup
                18. Jin S, Issel CJ, Montelaro RC. Serological method using recombinant S2 protein to differentiate equine infectious anemia virus (EIAV)-infected and EIAV-vaccinated horses.. Clin Diagn Lab Immunol 2004 Nov;11(6):1120-9.
                  doi: 10.1128/CDLI.11.6.1120-1129.2004pubmed: 15539516google scholar: lookup
                19. Chen C, Weisz OA, Stolz DB, Watkins SC, Montelaro RC. Differential effects of actin cytoskeleton dynamics on equine infectious anemia virus particle production.. J Virol 2004 Jan;78(2):882-91.
                  doi: 10.1128/jvi.78.2.882-891.2004pubmed: 14694119google scholar: lookup
                20. Chen C, Montelaro RC. Characterization of RNA elements that regulate gag-pol ribosomal frameshifting in equine infectious anemia virus.. J Virol 2003 Oct;77(19):10280-7.
                  doi: 10.1128/jvi.77.19.10280-10287.2003pubmed: 12970412google scholar: lookup
                21. Li F, Craigo JK, Howe L, Steckbeck JD, Cook S, Issel C, Montelaro RC. A live attenuated equine infectious anemia virus proviral vaccine with a modified S2 gene provides protection from detectable infection by intravenous virulent virus challenge of experimentally inoculated horses.. J Virol 2003 Jul;77(13):7244-53.
                  doi: 10.1128/jvi.77.13.7244-7253.2003pubmed: 12805423google scholar: lookup
                22. Howe L, Leroux C, Issel CJ, Montelaro RC. Equine infectious anemia virus envelope evolution in vivo during persistent infection progressively increases resistance to in vitro serum antibody neutralization as a dominant phenotype.. J Virol 2002 Nov;76(21):10588-97.
                  doi: 10.1128/jvi.76.21.10588-10597.2002pubmed: 12368301google scholar: lookup
                23. Li F, Chen C, Puffer BA, Montelaro RC. Functional replacement and positional dependence of homologous and heterologous L domains in equine infectious anemia virus replication.. J Virol 2002 Feb;76(4):1569-77.
                  doi: 10.1128/jvi.76.4.1569-1577.2002pubmed: 11799151google scholar: lookup
                24. Evans DT, Tillman KC, Desrosiers RC. Envelope glycoprotein cytoplasmic domains from diverse lentiviruses interact with the prenylated Rab acceptor.. J Virol 2002 Jan;76(1):327-37.
                  doi: 10.1128/jvi.76.1.327-337.2002pubmed: 11739697google scholar: lookup
                25. Chen C, Li F, Montelaro RC. Functional roles of equine infectious anemia virus Gag p9 in viral budding and infection.. J Virol 2001 Oct;75(20):9762-70.
                  doi: 10.1128/JVI.75.20.9762-9770.2001pubmed: 11559809google scholar: lookup
                26. Li F, Leroux C, Craigo JK, Cook SJ, Issel CJ, Montelaro RC. The S2 gene of equine infectious anemia virus is a highly conserved determinant of viral replication and virulence properties in experimentally infected ponies.. J Virol 2000 Jan;74(1):573-9.
                  doi: 10.1128/jvi.74.1.573-579.2000pubmed: 10590152google scholar: lookup
                27. Li F, Puffer BA, Montelaro RC. The S2 gene of equine infectious anemia virus is dispensable for viral replication in vitro.. J Virol 1998 Oct;72(10):8344-8.
                  doi: 10.1128/JVI.72.10.8344-8348.1998pubmed: 9733881google scholar: lookup
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