Rescue of Recombinant Newcastle Disease Virus from cDNA

Summary

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

Abstract

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae¹, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA^2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Introduction

Newcastle disease virus (NDV), an avian paramyxovirus belonging to the Avulavirus genus¹, is an economically relevant and thus widely researched and surveilled zoonotic agent, which can severely affect poultry farming all around the world. Although not a human pathogen, NDV has also been thoroughly studied beyond the veterinarian field both as a model paramyxovirus and due to its highly interesting, natural oncolytic properties⁶. Research on NDV greatly benefited from the development of reverse genetics techniques for single-stranded, non-segmented negative-sense RNA viruses, first described for rabies virus by Conzelmann and coleagues

Protocol

1. Preparation of Mammalian Cells (Figure 4A, Day 1)

Split HEp-2 or A549 cells the day before transfection in 6-well plates. Density of the cells should reach 80-90% confluence the following day. Usually, a confluent 100 mm dish can be split into 8 wells (around 1 x 10⁶ cells per well). For each virus to be rescued, 2-4 different wells should be included, as well as 2 extra wells for the controls pCAGGs-GFP and pCITE-GFP¹⁸, aimed to monitor transfection and MVA-T7 infection efficiencies, respectively.

2. Infection of Mammalian Cells with the Recombinant Modified Vaccinia Ankara ....

Results

Rescue of NDV is a well-established procedure, routinely performed in the laboratories that have access to the complete cDNA of the virus. However, the intrinsic stochastic nature of the method makes it difficult to achieve 100% rescue efficiency. Monitoring the early steps of the process, specially the transfection efficiency and the infection with MVA-T7, helps identifying possible problems. Figure 5A shows standard transfection and transfection/infection efficiencies that are enough for a successful N.......

Discussion

Several factors are to be considered to achieve good results while rescuing NDV. First, the full-length cDNA construct to be used needs to be designed to allow the functional incorporation of the new transgenes/modifications into the NDV genome. This means, as stated above, that (i) appropriate gene end (GE), intergenic (IG) and gene start (GS) sequences are to be added if required; (ii) there are no putative GE or GS sequences into the foreign gene, and (iii) the full recombinant genome follows the "rule of six".......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	CORNING Cellgro	10-013-CV	Any supplier
OptiMEM	GIBCO	31985-070
Lipofectamine 2000 (LPF2000)	Invitrogen	11668-019
35% Bovine Albumin (BA)	Sigma	232-936-2	Any supplier
Trypsin-EDTA	CORNING Cellgro	25-052-CI	Any supplier
Penicillin/Streptomycin (PS) 100x	CORNING Cellgro	30-002-CI	Any supplier
Fetal Bovine Serum (FBS)	Hyclone	SH30070.03	Any supplier
Cell lines A549 cells (catalogue number CRL-185), HEp-2 cells (catalogue number CRL-185), chicken embryo fibroblasts (catalogue number CRL-12203) and duck embryo fibroblasts (catalogue number CCL-141) are available from the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA. 20110-2209 USA). All cell lines are maintained in a 37 °C incubator with 5% CO2 in DMEM 10% FBS, 1% PS. Embryonated chicken eggs Embryonated chicken eggs can be obtained from Charles River Laboratories, Specific Pathogen Fee Avian Supply (SPAFAS) Avian Products and Services (Franklin Commons, 106 Route 32, North Franklin, CT 06254, USA) and are maintained at 37 °C. Viability of the embryos is assessed with an egg candler. Eggs are infected when they reach 8-10 days old. Both infection and harvest of the allantoic fluid takes place under sterile conditions. All eggs are autoclaved and discarded following standard laboratory biosafety protocols. Turkey red blood cells (RBC) Turkey RBC can be purchased from Truslow Farms (201 Valley Road, Chestertown, Md 21620, USA)and stored at 4 °C. To prepare RBC for HA assay, wash 5 ml of the commercial stock with 45 ml of PBS 1x in a 50 ml conical tube. Centrifuge for 5 min at 1,000 rpm and carefully discard the supernatant. Dilute pelleted RBC 1:1,000 in PBS 1x for a final 0.5-1.0% concentration. Washed RBC can be stored at 4 °C for several days. Plasmids Plasmid preparations are obtained with any commercially available maxiprep kit following manufacturer's instructions, diluted in ddH20 to a final concentration of 1 μg/μl and stored at -20 °C. DNA concentration and purity are assessed by spectrophotometry at 260 and 280 nm. Preparations with a 260/280 ratio higher than 1.8 are considered of acceptable quality for the rescue. Plasmid DNA quality is also routinely double-checked by agarose gel chromatography. Viruses The described protocol for rescue of the lentogenic NDV strain Hitchner B1 can be performed under biosafety level (BSL) 2 conditions. The Modified Vaccinia Ankara expressing the T7 RNA polymerase (MVA-T7) was described34 and obtained from Dr. Bernard Moss. This virus is growth in confluent monolayers of chicken embryo fibroblasts and titrated in mammalian (A549 or HEp-2) cells. NDV stocks are grown in embryonated chicken eggs and titrated by IFA using polyclonal serum raised against purified virions. All contaminated material should be safely sterilized and disposed according to standard biosafety procedures. Tissue culture media and solutions: DMEM 10% FBS 1% PS: Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and Penicillin/Streptomycin (P/S): 445 ml of DMEM, 50 ml of heat-inactivated FBS, 5 ml of 100x commercial P/S solution. Store at 4 °C. 10x Phosphate buffered saline (PBS): 80 g of NaCl, 2 g of KCl, 11.5 g of Na2HPO4.7H2O, 2 g of KH2PO4. Add ddH2O up to 1 L. Adjust pH to 7.3. Sterilize by autoclave. Store at room temperature. 1x PBS: Dilute 10x PBS 1:10 with ddH2O. Sterilize by autoclave and store at room temperature. 100x Ca/Mg : 1.327 g CaCl2.2H2O, 2.133 g MgCl2.6H2O and add ddH2O up to 100 ml. Autoclave and store at room temperature. 1x PBS/BA/PS: 50 ml of 10x Phosphate buffered saline (PBS) in 437 ml ddH2O. Autoclave and when cooled down to room temperature, add 5 ml 100x Penicillin/Streptomycin 3 ml 35% Bovine and 5 ml of 100x Ca/Mg. Store at 4 °C.