Reverse Genetics for Newcastle Disease Virus as a Vaccine Vector

Shin‐Hee Kim1, Siba K. Samal1

1 VA‐MD College of Veterinary Medicine, University of Maryland, College Park, Maryland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 18.5
DOI:  10.1002/cpmc.44
Online Posting Date:  February, 2018
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Newcastle disease virus (NDV) is an economically important pathogen in the poultry industry worldwide. Recovery of infectious NDV from cDNA using reverse genetics has made it possible to manipulate the genome of NDV. This has greatly contributed to our understanding of the molecular biology and pathogenesis of NDV. Furthermore, NDV has modular genome and accommodates insertion of a foreign gene as a transcriptional unit, thus enabling NDV as a vaccine vector against diseases of humans and animals. Avirulent NDV strains (e.g., LaSota and B1) have been commonly used as vaccine vectors. In this protocol, we have described reverse genetics of NDV to be used as a vaccine vector by exemplifying the recovery of NDV vectored avian influenza virus vaccine. Specifically, cloning and recovery of NDV expressing the hemagglutinin protein of highly pathogenic influenza virus were explained. © 2018 by John Wiley & Sons, Inc.

Keywords: avian influenza virus; newcastle disease virus; reverse genetics; vaccine vector

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Table of Contents

  • Introduction
  • Basic Protocol 1: Construction of Full‐Length NDV Containing a Protective Agent
  • Basic Protocol 2: Recovery of Recombinant NDV Expressing the HA Protein
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Construction of Full‐Length NDV Containing a Protective Agent

  • Synthesized HA gene (GenScript; sequence can be available upon the request)
  • Platinum Taq DNA polymerase (Invitrogen, cat. no. 10966026)
  • Primer set (forward and reverse, 10 µM stock concentration) (Table 18.5.1)
  • dNTPs (dATP, dCTP, dGTP, dTTP, 10 mM; see recipe)
  • Agarose‐TAE gel (1%; see recipe)
  • 6× DNA loading dye
  • Tris‐acetate‐EDTA (TAE) electrophoresis buffer (see recipe)
  • 1‐kb plus DNA ladder (i.e., Invitrogen and Fisher Scientific, cat. no. 10787018 and SM1331, respectively)
  • DNA purification kit (e.g., Qiagen QIAquick gel extraction, cat. no. 28706 and Clontech NucleoSpin Gel and PCR Clean‐up, cat. no. 740609)
  • k PmeI restriction enzyme (New England Biolabs, cat. no.R0560L)
  • Midi plasmid purification kit (e.g., QIAGEN Plasmid Midi Purification, cat. no. 12145 and Clontech NucleoBond Xtra Midi, cat. no 740410)Mini plasmid preparation kit (e.g., QIAprep Spin Miniprep, cat. no. 27106 and NucleoSpin Plasmid, cat. no. 740499)
  • Alkaline phosphatase (e.g., Calf Intestinal and shrimp, cat. nos. M0290S and M0371S, respectively)
  • Ligase (New England Biolabs, cat. no. M0202L)
  • E. coli (DH10B) chemically competent cells
  • Ice
  • Low‐salt LB agar plate supplemented with 5 μg/ml tetracycline (see reciperecipes)
  • Low‐salt LB broth supplemented with 5 μg/ml tetracycline (see reciperecipes)
Table 8.5.1   MaterialsPrimers used for construction of vaccine virus and DNA sequencing analysis

Primer a Sequence

 aF: Forward primer; R: Reverse primer
 bPmeI site is italicized and the NDV GS and GE signals are underlined.
  • PCR tubes (0.2 ml)
  • Thermal cycler
  • UV transilluminator
  • Razor blades
  • 1.5‐ml microcentrifuge tubes
  • Spectrophotometer(e.g., NanoDrop, Thermo Fisher Scientific)
  • Water bath (42°C)
  • Incubator (30°C)
  • Rocking incubator (30°C)
  • Sterile 15‐ml round‐bottom culture tubes (e.g., BD)

Basic Protocol 2: Recovery of Recombinant NDV Expressing the HA Protein

  • Human epidermoid carcinoma cell line (HEp‐2)
  • Plasmids (pLaSota‐HA, pTM1‐N, pTM1‐P, and pTM1‐L)
  • Opti‐MEM medium (Gibco, cat. no. 31985070)
  • Transfection agent (e.g., Lipofectamine 2000, Invitrogen, cat. no. 11668019 and PolyJet™ In Vitro DNA, SignaGen Laboratories, cat. no. SL100688)
  • Modified vaccinia virus Ankara (MVA; BEI Resources, cat. no. NR‐1)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Dulbecco's Modified Eagle Medium (DMEM; Gibco, cat. no. 11966‐025)
  • Penicillin/streptomycin solution (100×; Gibco, cat. no. 15140122)
  • Freshly collected allantoic fluid from 10‐ to 11‐day‐old embryonated chicken eggs (store up to 1 week at 4°C)
  • 9‐ to 10‐day‐old specific pathogen‐free (SPF) embryonated chicken eggs (Charles River)
  • Chicken embryo fibroblast cell line (DF1)
  • 70% ethanol
  • 1% chicken red blood cells (RBC; Rampine Laboratories, cat. no. 7401203)
  • Methylcellulose medium (see recipe)
  • Fetal Bovine Serum (FBS; Gibco, cat. no. 10437028)
  • Methanol
  • 1% crystal violate (see recipe)
  • Biosafety cabinet class II
  • 6‐well tissue culture plate
  • 37°C, 5% CO 2 humidified incubator
  • Metal spoon
  • Forceps
  • Disposable spoon (Fisher, cat. no. 14‐375‐255)
  • Conical centrifuge tube (15 ml)
  • Centrifuge
  • V‐bottom 96‐well plates
  • Vortex mixer
  • Micropipette tips, sterile
  • 1.5‐ml microcentrifuge tubes
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Literature Cited

Literature Cited
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