Generation of Newcastle Disease Virus (NDV) Recombinants Expressing the Infectious Laryngotracheitis Virus (ILTV) Glycop
Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens caused by infection with infectious laryngotracheitis virus (ILTV), a member of the family Herpesviridae. The current commercial ILT vaccines are either unsafe
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Introduction Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens that has become a major problem in the US poultry industry in recent years [1]. The disease is controlled mainly through biosecurity and vaccination with live attenuated strains of infectious laryngotracheitis virus (ILTV) and vectored vaccines based on turkey herpesvirus (HVT) and fowlpox virus (FPV) [2–5]. The current live attenuated vaccines (chicken embryo origin [CEO] and tissue culture origin [TCO]), although effective, can regain virulence. ILT vaccines based on HVT and FPV vectors are less efficacious than live attenuated vaccines [5–7]. Therefore, there is a pressing need to develop safer and more efficacious ILT vaccines. Newcastle disease virus (NDV) is the causative agent of Newcastle disease (ND), one of the most important poultry diseases worldwide, affecting a wide variety of birds and causing significant economic losses to the poultry industry [8]. The NDV LaSota strain, a naturally occurring low-virulence NDV strain, has been routinely used as a live vaccine throughout the world [9]. This vaccine strain induces strong immunity both locally and systemically and can be readily administered through drinking water supplies or by direct spray [10]. For the last 60 years, the LaSota vaccine has been proven to be safe and stable, with no reports of reversion to virulence or recombination with field strains. During the past decade, the LaSota vaccine and other NDV strains have been developed as vectors using reverse genetics technology in order to express foreign genes for vaccine or gene therapy purposes [11–13].
Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 2: Vaccines for Veterinary Diseases, Methods in Molecular Biology, vol. 1404, DOI 10.1007/978-1-4939-3389-1_6, © Springer Science+Business Media New York 2016
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Wei Zhao et al.
Here, we describe the strategy and protocol for the construction of NDV LaSota vaccine strain-based cDNA clones vectoring the glycoprotein genes (gB and gD) of ILTV and the rescue of infectious NDV recombinants from cloned cDNAs, as dual vaccines against ILT and ND using reverse genetics technology.
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Materials
2.1 Cloning ILTV gB and gD Genes
1. The ILTV strain (63140/C/08/BR) [14]. 2. 10 mM deoxynucleotide triphosphate (dNTP) mix (New England Biolabs, Ipswich, MA). 3. Primers used for cloning: Plant-gB/GFP F: 5′-ATAGTTGTAGCACCATGCAATCCTA CATCG-3′. Plant-gB/GFP R: 5′-GTAGTTACACACAGCTTATTCGTCT TCGCTTTC-3′. Plant-gD/GFP F: 5′-ATAGTTGTAGCACCATGCACCGTCC TCATC-3′. Plant-gD/GFP R: 5′-GTAGTTACACACAGCTTAGCTACG CGCGCAT-3′. 4. PfuUltra II fusion HS DNA polymerase (Agilent Technologies, La Jolla, CA). 5. Agarose. 6. 1× TAE buffer. 7. 1 kb Plus DNA Ladder (Life Technologies, Carlsbad, CA). 8. SYBR® Safe DNA gel stain (Life Technologies). 9. QIAquick gel extraction kit (Qiagen, Valencia, CA). 10. Isopropanol.
2.2 Preparation of a Linearized NDV Vector
1. The pLS-GFP plasmid [15]. 2. Primers used for linearizing NDV vector: Insert vec up: 5′-GGT
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