FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / J Vet Med B Infect Dis Vet Public Health / Cloning of the genomes of equine herpesvirus type 1 (EHV-1) ...
Journal of veterinary medicine. B, Infectious diseases and veterinary public health2002; 49(1); 31-36; doi: 10.1046/j.1439-0450.2002.00534.x

Cloning of the genomes of equine herpesvirus type 1 (EHV-1) strains KyA and racL11 as bacterial artificial chromosomes (BAC).

Abstract: The genome of equine herpesvirus type 1 (EHV-1) strain RacL11, a highly virulent isolate obtained from an aborted foal, and that of the modified live vaccine strain KyA, were cloned as bacterial artificial chromosomes (BAC) in Eseherichia coli. Mini F plasmid sequences were inserted into the viral genomes by homologous recombination instead of the gene 71 (EUS4) open reading frame after co-transfection of viral DNA and recombinant plasmid pdelta71-pHA2 into RK13 cells. After isolation of recombinant viruses by three rounds of plaque purification, viral DNA was isolated from RK13 cells infected with RacL11 or KyA virus mutants expressing the green fluorescent protein (GFP), and electroporated into Escherichia coli DH10B cells. Several bacterial colonies were shown to contain high-molecular weight BAC DNA with a restriction enzyme fragment pattern indicative of the presence of full-length RacL11 or KyA genomes. Two selected BAC clones were analysed by restriction enzyme analysis and Southern blotting, and were eventually termed pRacLI I and pKyA. respectively. Upon transfection of pRacL11 or pKyA DNA into RK13 cells, GFP-expressing fluorescing virus plaques could be identified from day 1 after transfection. Infectivity after transfection of pRacL11 or pKyA could be readily propagated on RK13 or equine cells, indicating that infectious full-length DNA clones of strains RacL11 and KyA were successfully cloned in Escherichia coli as BACs. The glycoprotein 2-negative progeny reconstituted from pRacL11 and pKyA (L11deltagp2 and KyAdeltagp2) exhibited different growth properties. Whereas both L11deltagp2 and KyAdeltagp2 extracellular titres were reduced by 15- to 32-fold, plaque diameters were only markedly (50%) reduced in the case of KyAdeltagp2.
Get Full Text
Publication Date: 2002-03-26 PubMed ID: 11911590DOI: 10.1046/j.1439-0450.2002.00534.xGoogle Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article
  • Research Support
  • Non-U.S. Gov't
  • Research Support
  • U.S. Gov't
  • P.H.S.

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 paper focuses on the successful cloning of the genomes of equine herpesvirus type 1 (EHV-1) strains KyA and racL11 using a technique involving bacterial artificial chromosomes (BACs) in E.coli. The results of this study have potential implications for the development of vaccines and diagnostic tests for equine herpesviruses.

Introduction and Methods

  • The researchers commenced the study by focusing on two strains of EHV-1. One of the strains was RacL11, a highly virulent virus replicating at high rates, and the other was KyA, a modified live vaccine strain.
  • Their genomes were cloned into bacterial artificial chromosomes (BACs) in Escherichia coli. BACs are DNA sequences that bacteria can replicate and, in this case, were used as vectors (carriers) for the viral genome.
  • A specific piece of plasmid DNA sequence was inserted into the viral genome through a process known as homologous recombination, replacing the EUS4 gene (gene 71).

Study and Results

  • The recombined viruses underwent three rounds of plaque purification. Plaque purification is a method used to isolate a pure strain of virus particles. The recombinant viruses were then extracted from infected cells.
  • The isolated viruses, now emitting a green fluorescent protein (GFP), were transferred into Escherichia coli (bacteria) cells. The introduction of high-molecular weight BAC DNA into the bacteria resulted in bacterial colonies carrying full-length RacL11 or KyA genomes.
  • Two selected BAC clones were named pRacL11 and pKyA after analysis via restriction enzyme analysis and Southern blotting, techniques used to identify specific sequences within DNA or RNA samples.

Findings

  • Following this, the pRacL11 and pKyA were reintroduced into cells, displaying green fluorescent protein (GFP) identifying virus plaques.
  • The transfected viruses were easily propagated (multiplied) in the host cells, thereby confirming the successful cloning of the full-length DNA clones of the RacL11 and KyA strains in E.coli as BACs.
  • However, when the viral strains lacking the glycoprotein 2 gene were analysed, they displayed different growth properties. Both had reduced extracellular viral titres (amount of virus in an infected organism), but the size of plaques (areas of infection) were significantly noted only in the KyAdeltagp2 strain.

Cite This Article

APA
Rudolph J, O'Callaghan DJ, Osterrieder N. (2002). Cloning of the genomes of equine herpesvirus type 1 (EHV-1) strains KyA and racL11 as bacterial artificial chromosomes (BAC). J Vet Med B Infect Dis Vet Public Health, 49(1), 31-36. https://doi.org/10.1046/j.1439-0450.2002.00534.x

Publication

Journal of veterinary medicine. B, Infectious diseases and veterinary public health
ISSN: 0931-1793
NlmUniqueID: 100955260
Country: Germany
Language: English
Volume: 49
Issue: 1
Pages: 31-36

Researcher Affiliations

Rudolph, J
  • Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, Insel Riems, Germany.
O'Callaghan, D J
    Osterrieder, N

      MeSH Terms

      • Animals
      • Chromosomes, Artificial, Bacterial / genetics
      • Cloning, Molecular
      • DNA Primers
      • DNA, Viral / genetics
      • Escherichia coli / genetics
      • Fluorescent Antibody Technique
      • Genome, Viral
      • Herpesviridae Infections / veterinary
      • Herpesviridae Infections / virology
      • Herpesvirus 1, Equid / classification
      • Herpesvirus 1, Equid / genetics
      • Herpesvirus 1, Equid / pathogenicity
      • Horse Diseases / virology
      • Horses
      • Mutagenesis
      • Plasmids / genetics
      • Polymerase Chain Reaction / veterinary
      • Transfection / veterinary
      • Vaccines, Attenuated / genetics
      • Virulence

      Grant Funding

      • AI22001 / NIAID NIH HHS

      Citations

      This article has been cited 32 times.
      1. Kremling V, Loll B, Pach S, Dahmani I, Weise C, Wolber G, Chiantia S, Wahl MC, Osterrieder N, Azab W. Crystal structures of glycoprotein D of equine alphaherpesviruses reveal potential binding sites to the entry receptor MHC-I. Front Microbiol 2023;14:1197120.
        doi: 10.3389/fmicb.2023.1197120pubmed: 37250020google scholar: lookup
      2. Vissani MA, Damiani AM, Barrandeguy ME. Equine Coital Exanthema: New Insights on the Knowledge and Leading Perspectives for Treatment and Prevention. Pathogens 2021 Aug 20;10(8).
        doi: 10.3390/pathogens10081055pubmed: 34451519google scholar: lookup
      3. Kolyvushko O, Latzke J, Dahmani I, Osterrieder N, Chiantia S, Azab W. Differentially-Charged Liposomes Interact with Alphaherpesviruses and Interfere with Virus Entry. Pathogens 2020 May 8;9(5).
        doi: 10.3390/pathogens9050359pubmed: 32397270google scholar: lookup
      4. Van Cleemput J, Poelaert KCK, Laval K, Vanderheijden N, Dhaenens M, Daled S, Boyen F, Pasmans F, Nauwynck HJ. An Alphaherpesvirus Exploits Antimicrobial β-Defensins To Initiate Respiratory Tract Infection. J Virol 2020 Mar 31;94(8).
        doi: 10.1128/JVI.01676-19pubmed: 31996426google scholar: lookup
      5. Wimer CL, Schnabel CL, Perkins G, Babasyan S, Freer H, Stout AE, Rollins A, Osterrieder N, Goodman LB, Glaser A, Wagner B. The deletion of the ORF1 and ORF71 genes reduces virulence of the neuropathogenic EHV-1 strain Ab4 without compromising host immunity in horses. PLoS One 2018;13(11):e0206679.
        doi: 10.1371/journal.pone.0206679pubmed: 30440016google scholar: lookup
      6. Wang J, Guo R, Qiao Y, Xu M, Wang Z, Liu Y, Gu Y, Liu C, Hou J. An inactivated gE-deleted pseudorabies vaccine provides complete clinical protection and reduces virus shedding against challenge by a Chinese pseudorabies variant. BMC Vet Res 2016 Dec 7;12(1):277.
        doi: 10.1186/s12917-016-0897-zpubmed: 27923365google scholar: lookup
      7. Proft A, Spiesschaert B, Izume S, Taferner S, Lehmann MJ, Azab W. The Role of the Equine Herpesvirus Type 1 (EHV-1) US3-Encoded Protein Kinase in Actin Reorganization and Nuclear Egress. Viruses 2016 Oct 12;8(10).
        doi: 10.3390/v8100275pubmed: 27754319google scholar: lookup
      8. Azab W, Gramatica A, Herrmann A, Osterrieder N. Binding of alphaherpesvirus glycoprotein H to surface α4β1-integrins activates calcium-signaling pathways and induces phosphatidylserine exposure on the plasma membrane. mBio 2015 Oct 20;6(5):e01552-15.
        doi: 10.1128/mBio.01552-15pubmed: 26489864google scholar: lookup
      9. Spiesschaert B, Osterrieder N, Azab W. Comparative analysis of glycoprotein B (gB) of equine herpesvirus type 1 and type 4 (EHV-1 and EHV-4) in cellular tropism and cell-to-cell transmission. Viruses 2015 Feb 3;7(2):522-42.
        doi: 10.3390/v7020522pubmed: 25654240google scholar: lookup
      10. Azab W, Lehmann MJ, Osterrieder N. Glycoprotein H and α4β1 integrins determine the entry pathway of alphaherpesviruses. J Virol 2013 May;87(10):5937-48.
        doi: 10.1128/JVI.03522-12pubmed: 23514881google scholar: lookup
      11. Azab W, Zajic L, Osterrieder N. The role of glycoprotein H of equine herpesviruses 1 and 4 (EHV-1 and EHV-4) in cellular host range and integrin binding. Vet Res 2012 Aug 21;43(1):61.
        doi: 10.1186/1297-9716-43-61pubmed: 22909178google scholar: lookup
      12. Charvat RA, Zhang Y, O'Callaghan DJ. Deletion of the UL4 gene sequence of equine herpesvirus 1 precludes the generation of defective interfering particles. Virus Genes 2012 Oct;45(2):295-303.
        doi: 10.1007/s11262-012-0781-2pubmed: 22752566google scholar: lookup
      13. Tischer BK, Kaufer BB. Viral bacterial artificial chromosomes: generation, mutagenesis, and removal of mini-F sequences. J Biomed Biotechnol 2012;2012:472537.
        doi: 10.1155/2012/472537pubmed: 22496607google scholar: lookup
      14. Hall RN, Meers J, Fowler E, Mahony T. Back to BAC: the use of infectious clone technologies for viral mutagenesis. Viruses 2012 Feb;4(2):211-35.
        doi: 10.3390/v4020211pubmed: 22470833google scholar: lookup
      15. Dai G, Kim S, O'Callaghan DJ, Kim SK. Development of a bacterial artificial chromosome (BAC) recombineering procedure using galK-untranslated region (UTR) for the mutation of diploid genes. J Virol Methods 2012 Jun;182(1-2):18-26.
        doi: 10.1016/j.jviromet.2012.02.010pubmed: 22407056google scholar: lookup
      16. Ma G, Feineis S, Osterrieder N, Van de Walle GR. Identification and characterization of equine herpesvirus type 1 pUL56 and its role in virus-induced downregulation of major histocompatibility complex class I. J Virol 2012 Apr;86(7):3554-63.
        doi: 10.1128/JVI.06994-11pubmed: 22278226google scholar: lookup
      17. Azab W, Osterrieder N. Glycoproteins D of equine herpesvirus type 1 (EHV-1) and EHV-4 determine cellular tropism independently of integrins. J Virol 2012 Feb;86(4):2031-44.
        doi: 10.1128/JVI.06555-11pubmed: 22171258google scholar: lookup
      18. Ahn BC, Kim S, Zhang Y, Charvat RA, O'Callaghan DJ. The early UL3 gene of equine herpesvirus-1 encodes a tegument protein not essential for replication or virulence in the mouse. Virology 2011 Nov 10;420(1):20-31.
        doi: 10.1016/j.virol.2011.08.016pubmed: 21917286google scholar: lookup
      19. Charvat RA, Breitenbach JE, Ahn B, Zhang Y, O'Callaghan DJ. The UL4 protein of equine herpesvirus 1 is not essential for replication or pathogenesis and inhibits gene expression controlled by viral and heterologous promoters. Virology 2011 Apr 10;412(2):366-77.
        doi: 10.1016/j.virol.2011.01.025pubmed: 21324502google scholar: lookup
      20. Zhou F, Gao SJ. Recent advances in cloning herpesviral genomes as infectious bacterial artificial chromosomes. Cell Cycle 2011 Feb 1;10(3):434-40.
        doi: 10.4161/cc.10.3.14708pubmed: 21245660google scholar: lookup
      21. Ahn B, Zhang Y, Osterrieder N, O'Callaghan DJ. Properties of an equine herpesvirus 1 mutant devoid of the internal inverted repeat sequence of the genomic short region. Virology 2011 Feb 20;410(2):327-35.
        doi: 10.1016/j.virol.2010.11.020pubmed: 21176938google scholar: lookup
      22. Warden C, Tang Q, Zhu H. Herpesvirus BACs: past, present, and future. J Biomed Biotechnol 2011;2011:124595.
        doi: 10.1155/2011/124595pubmed: 21048927google scholar: lookup
      23. Hofmann-Sieber H, Wild J, Fiedler N, Tischer K, von Einem J, Osterrieder N, Hofmann H, Köstler J, Wagner R. Impact of ETIF deletion on safety and immunogenicity of equine herpesvirus type 1-vectored vaccines. J Virol 2010 Nov;84(22):11602-13.
        doi: 10.1128/JVI.00677-10pubmed: 20826695google scholar: lookup
      24. Ahn BC, Zhang Y, O'Callaghan DJ. The equine herpesvirus-1 (EHV-1) IR3 transcript downregulates expression of the IE gene and the absence of IR3 gene expression alters EHV-1 biological properties and virulence. Virology 2010 Jul 5;402(2):327-37.
        doi: 10.1016/j.virol.2010.03.051pubmed: 20417949google scholar: lookup
      25. Breitenbach JE, Ebner PD, O'Callaghan DJ. The IR4 auxiliary regulatory protein expands the in vitro host range of equine herpesvirus 1 and is essential for pathogenesis in the murine model. Virology 2009 Jan 20;383(2):188-94.
        doi: 10.1016/j.virol.2008.10.017pubmed: 19012943google scholar: lookup
      26. Hain T, Otten S, von Both U, Chatterjee SS, Technow U, Billion A, Ghai R, Mohamed W, Domann E, Chakraborty T. Novel bacterial artificial chromosome vector pUvBBAC for use in studies of the functional genomics of Listeria spp. Appl Environ Microbiol 2008 Mar;74(6):1892-901.
        doi: 10.1128/AEM.00415-07pubmed: 18223114google scholar: lookup
      27. von Einem J, Schumacher D, O'Callaghan DJ, Osterrieder N. The alpha-TIF (VP16) homologue (ETIF) of equine herpesvirus 1 is essential for secondary envelopment and virus egress. J Virol 2006 Mar;80(6):2609-20.
        doi: 10.1128/JVI.80.6.2609-2620.2006pubmed: 16501071google scholar: lookup
      28. Guggemoos S, Just FT, Neubauer A. The equine herpesvirus 1 UL20 product interacts with glycoprotein K and promotes egress of mature particles. J Virol 2006 Jan;80(1):95-107.
        doi: 10.1128/JVI.80.1.95-107.2006pubmed: 16352534google scholar: lookup
      29. Trapp S, von Einem J, Hofmann H, Köstler J, Wild J, Wagner R, Beer M, Osterrieder N. Potential of equine herpesvirus 1 as a vector for immunization. J Virol 2005 May;79(9):5445-54.
        doi: 10.1128/JVI.79.9.5445-5454.2005pubmed: 15827159google scholar: lookup
      30. Smith PM, Kahan SM, Rorex CB, von Einem J, Osterrieder N, O'Callaghan DJ. Expression of the full-length form of gp2 of equine herpesvirus 1 (EHV-1) completely restores respiratory virulence to the attenuated EHV-1 strain KyA in CBA mice. J Virol 2005 Apr;79(8):5105-15.
        doi: 10.1128/JVI.79.8.5105-5115.2005pubmed: 15795295google scholar: lookup
      31. Redwood AJ, Messerle M, Harvey NL, Hardy CM, Koszinowski UH, Lawson MA, Shellam GR. Use of a murine cytomegalovirus K181-derived bacterial artificial chromosome as a vaccine vector for immunocontraception. J Virol 2005 Mar;79(5):2998-3008.
        doi: 10.1128/JVI.79.5.2998-3008.2005pubmed: 15709020google scholar: lookup
      32. von Einem J, Wellington J, Whalley JM, Osterrieder K, O'Callaghan DJ, Osterrieder N. The truncated form of glycoprotein gp2 of equine herpesvirus 1 (EHV-1) vaccine strain KyA is not functionally equivalent to full-length gp2 encoded by EHV-1 wild-type strain RacL11. J Virol 2004 Mar;78(6):3003-13.
        doi: 10.1128/jvi.78.6.3003-3013.2004pubmed: 14990719google scholar: lookup
      Subscribe to our newsletter
      Join 99,383 horse owners like you who receive our email updates on horse health and nutrition.