Abstract
Equine herpesvirus 4 (EHV-4) is a major cause of respiratory tract disease in horses worldwide. The generation of recombinant viruses, which would lead to understanding of viral gene functions, has been hindered by the absence of suitable cell lines and small-animal models of the infection. In the present study, the genome of EHV-4 strain TH20p was cloned as a stable and infectious BAC without any deletions of the viral genes. Mini F plasmid sequences flanked by loxP sites were inserted into the intergenic region between genes 58 and 59. Coinfection of the recombinant virus with a recombinant adenovirus expressing Cre recombinase resulted in the excision of the BAC sequences. Importantly, the resulting recombinant EHV-4 replicated comparably to the wild-type virus in fetal horse kidney cells. The recombinant EHV-4 will facilitate EHV-4 research and provide the opportunity to exploit the power of BAC technology for production of recombinant viral vaccines.
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References
Adler H, Messerle M, Wagner M, Koszinowski UH (2000) Cloning and mutagenesis of the murine gammaherpesvirus 68 genome as an infectious bacterial artificial chromosome. J Virol 74:6964–6974
Adler H, Messerle M, Koszinowski UH (2003) Cloning of herpesviral genomes as bacterial artificial chromosomes. Rev Med Virol 13:111–121
Arii J, Hushur O, Kato K, Kawaguchi Y, Tohya Y, Akashi H (2006) Construction of an infectious clone of canine herpesvirus genome as a bacterial artificial chromosome. Microbes Infect 8:1054–1063
Azmi M, Field HJ (1993) Interactions between equine herpesvirus type 1 and equine herpesvirus type 4: T cell responses in a murine infection model. J Gen Virol 74:2339–2345
Borst EM, Hahn G, Koszinowski UH, Messerle M (1999) Cloning of the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli: a new approach for construction of HCMV mutants. J Virol 73:8320–8329
Chang WL, Barry PA (2003) Cloning of the full-length rhesus cytomegalovirus genome as an infectious and self-excisable bacterial artificial chromosome for analysis of viral pathogenesis. J Virol 77:5073–5083
Damiani AM, Jang HK, Matsumura T, Yokoyama N, Miyazawa T, Mikami T (1999) Transcriptional analyses of the region of the equine herpesvirus type 4 genome encoding glycoproteins I and E. Arch Virol 144:613–620
Dewals B, Boudry C, Gillet L, Markine-Goriaynoff N, de Leval L, Haig DM, Vanderplasschen A (2006) Cloning of the genome of Alcelaphine herpesvirus 1 as an infectious and pathogenic bacterial artificial chromosome. J Gen Virol 87:509–517
Hansen K, Napier I, Koen M, Bradford S, Messerle M, Bell E, Seshadri L, Stokes HW, Birch D, Whalley JM (2006) In vitro transposon mutagenesis of an equine herpesvirus 1 genome cloned as a bacterial artificial chromosome. Arch Virol 151:2389–2405
Heintz N (2001) BAC to the future: the use of bac transgenic mice for neuroscience research. Nat Rev Neurosci 2:861–870
Hirt B (1967) Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol 26:365–369
Horsburgh BC, Hubinette MM, Qiang D, MacDonald ML, Tufaro F (1999a) Allele replacement: an application that permits rapid manipulation of herpes simplex virus type 1 genomes. Gene Ther 6:922–930
Horsburgh BC, Hubinette MM, Tufaro F (1999b) Genetic manipulation of herpes simplex virus using bacterial artificial chromosomes. Methods Enzymol 306:337–352
Kanegae Y, Lee G, Sato Y, Tanaka M, Nakai M, Sakaki T, Sugano S, Saito I (1995) Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase. Nucleic Acids Res 23:3816–3821
Kawakami Y, Kaji T, Ishizaki R, Shimizu T, Matumoto M (1962) Etiologic study on an outbreak of acute respiratory disease among colts due to equine rhinopneumonitis virus. Jpn J Exp Med 32:211–229
Maeda K, Mizukoshi F, Hamano M, Kai K, Iwata H, Kondo T, Matsumura T (2004) Development of an equine herpesvirus type 4-specific enzyme-linked immunosorbent assay using a B-cell epitope as an antigen. J Clin Microbiol 42:1095–1098
Maeda K, Kai K, Matsumura T (2005) Genomic diversity among equine herpesvirus-4 field isolates. J Vet Med Sci 67:555–561
Maeda K, Yasumoto S, Tsuruda A, Andoh K, Kai K, Otoi T, Matsumura T (2007) Establishment of a novel equine cell line for isolation and propagation of equine herpesviruses. J Vet Med Sci 69:989–991
Mahony TJ, McCarthy FM, Gravel JL, West L, Young PL (2002) Construction and manipulation of an infectious clone of the bovine herpesvirus 1 genome maintained as a bacterial artificial chromosome. J Virol 76:6660–6668
Matsumura T, Sugiura T, Imagawa H, Fukunaga Y, Kamada M (1992) Epizootiological aspects of type 1 and type 4 equine herpesvirus infections among horse populations. J Vet Med Sci 54:207–211
McGregor A, Schleiss MR (2001) Molecular cloning of the guinea pig cytomegalovirus (GPCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli. Mol Genet Metab 72:15–26
Messerle M, Crnkovic I, Hammerschmidt W, Ziegler H, Koszinowski UH (1997) Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci USA 94:14759–14763
Mizukoshi F, Maeda K, Hamano M, Iwata H, Matsumura T, Kondo T, Sugiura T (2002) IgG antibody subclass response against equine herpesvirus type 4 in horses. Vet Immunol Immunopathol 88:97–101
Nagaike K, Mori Y, Gomi Y, Yoshii H, Takahashi M, Wagner M, Koszinowski U, Yamanishi K (2004) Cloning of the varicella-zoster virus genome as an infectious bacterial artificial chromosome in Escherichia coli. Vaccine 22:4069–4074
Palfi V, Christensen LS (1995) Analyses of restriction fragment patterns (RFPs) and pathogenicity in baby mice of equine herpesvirus 1 and 4 (EHV-1 and EHV-4) strains circulating in Danish horses. Vet Microbiol 47:199–204
Patel JR, Heldens J (2005) Equine herpesviruses 1 (EHV-1) and 4 (EHV-4)—epidemiology, disease and immunoprophylaxis: a brief review. Vet J 170:14–23
Reed SM, Toribio RE (2004) Equine herpesvirus 1 and 4. Vet Clin North Am Equine Pract 20:631–642
Roizman B (1996) Herpesviridae. In: Field BN, Knipe DM, Howley PM, Channock RM, Melnick JL, Monath TP, Roizman B, Straus SE (eds) Virology, 3rd edn. Lippincott-Raven, Philadelphia
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 49:31–36
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor
Shizuya H, Kouros-Mehr H (2001) The development and applications of the bacterial artificial chromosome cloning system. Keio J Med 50:26–30
Sinzger C, Knapp J, Schmidt K, Kahl M, Jahn G (1999) A simple and rapid method for preparation of viral DNA from cell associated cytomegalovirus. J Virol Methods 81:115–122
Smith GA, Enquist LW (1999) Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus, an alphaherpesvirus. J Virol 73:6405–6414
Tanaka M, Kagawa H, Yamanashi Y, Sata T, Kawaguchi Y (2003) Construction of an excisable bacterial artificial chromosome containing a full-length infectious clone of herpes simplex virus type 1: viruses reconstituted from the clone exhibit wild-type properties in vitro and in vivo. J Virol 77:1382–1391
Telford EA, Watson MS, McBride K, Davison AJ (1992) The DNA sequence of equine herpesvirus-1. Virology 189:304–316
Telford EA, Watson MS, Perry J, Cullinane AA, Davison AJ (1998) The DNA sequence of equine herpesvirus-4. J Gen Virol 79:1197–1203
Tischer BK, Kaufer BB, Sommer M, Wussow F, Arvin AM, Osterrieder N (2007) A self-excisable infectious bacterial artificial chromosome clone of varicella-zoster virus allows analysis of the essential tegument protein encoded by ORF9. J Virol 81:13200–13208
Trapp S, Osterrieder N, Keil GM, Beer M (2003) Mutagenesis of a bovine herpesvirus type 1 genome cloned as an infectious bacterial artificial chromosome: analysis of glycoprotein E and G double deletion mutants. J Gen Virol 84:301–306
Wagner M, Ruzsics Z, Koszinowski UH (2002) Herpesvirus genetics has come of age. Trends Microbiol 10:318–324
Whalley JM, Ruitenberg KM, Sullivan K, Seshadri L, Hansen K, Birch D, Gilkerson JR, Wellington JE (2007) Host cell tropism of equine herpesviruses: glycoprotein D of EHV-1 enables EHV-4 to infect a non-permissive cell line. Arch Virol 152:717–725
Yu D, Smith GA, Enquist LW, Shenk T (2002) Construction of a self-excisable bacterial artificial chromosome containing the human cytomegalovirus genome and mutagenesis of the diploid TRL/IRL13 gene. J Virol 76:2316–2328
Zhang Z, Rowe J, Wang W, Sommer M, Arvin A, Moffat J, Zhu H (2007) Genetic analysis of varicella-zoster virus ORF0 to ORF4 by use of a novel luciferase bacterial artificial chromosome system. J Virol 81:9024–9033
Zhang Z, Huang Y, Zhu H (2008) A highly efficient protocol of generating and analyzing VZV ORF deletion mutants based on a newly developed luciferase VZV BAC system. J Virol Methods 148:197–204
Zhou FC, Zhang YJ, Deng JH, Wang XP, Pan HY, Hettler E, Gao SJ (2002) Efficient infection by a recombinant Kaposi’s sarcoma-associated herpesvirus cloned in a bacterial artificial chromosome: application for genetic analysis. J Virol 76:6185–6196
Acknowledgments
The authors are grateful to Dr. A. Vanderplasschen, University of Liege, and Dr. N. Osterrieder, Cornell University, for useful discussions and technical advice. This work was supported in part by grants from the Egyptian Ministry of Higher Education and a Grant-in-Aid for Scientific Research in Priority Areas from the Ministry of Education, Culture, Science, Sports and Technology (MEXT) of Japan.
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Azab, W., Kato, K., Arii, J. et al. Cloning of the genome of equine herpesvirus 4 strain TH20p as an infectious bacterial artificial chromosome. Arch Virol 154, 833–842 (2009). https://doi.org/10.1007/s00705-009-0382-0
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DOI: https://doi.org/10.1007/s00705-009-0382-0