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 coleagues². A variety of genetically modified NDVs, carrying foreign genes or modifications of their wild type genome have been widely studied ever since. Work with these recombinant viruses has been critical to characterize different virulence factors not only of NDV but also of other relevant human pathogens such as influenza A virus⁷ - or the emergent Nipah virus⁸. Furthermore, a number of different studies have explored the use of these techniques to improve the innate antitumoral activity of NDV^6,9,10, mostly by enhancing the immunostimulatory properties of the virus. Other relevant area of research on recombinant NDVs has been the generation of vaccine candidates against other viral diseases such as influenza^5,11,12, HIV¹³, measles¹⁴, SARS¹⁵, or that caused by the respiratory sincytial virus (RSV)¹⁶. Amongst the various noteworthy advantages provided by NDV are the lack of preexisting immunity in human populations, the stability of the foreign genetic inserts, a lack of recombinatory activity and overall a high safety profile combined with the aforementioned natural immunostimulatory properties¹⁷. It's also noteworthy the potential use of recombinant bivalent vaccines in poultry, protective against both NDV and highly pathogenic avian influenza viruses^11,12. This may be an excellent way to decrease the chances of the latter spreading from wild to domesticated animals, thus also helping to prevent a possible inter-specific jump of the dreaded avian influenza to humans. Finally, reporter-expressing NDV have been used for the evaluation of innate immune responses as well as the identification of interferon antagonist encoded by multiple viruses^18-27.

The rescue process of a recombinant, non-segmented, negative-stranded RNA virus basically consists on artificially forcing a viral replication cycle in a producing cell by transfecting cDNA encoding the minimal infective molecular machinery, known as ribonucleoprotein or RNP (Figure 2). The RNPs consist of the viral polymerase (P and L proteins), the nucleoprotein (NP) and the full-length antigenomic RNA of the virus. This RNA+ antigenome is the template required for the generation of the complementary RNA- genomes, which, also associated with the rest of the proteins of the viral RNP, recapitulates the same infectious complex that a natural virus would release on the cytoplasm of the cell upon infection (Figure 2A). From this step onwards, the viral cycle can proceed naturally and recombinant virions, encapsidating the modified genomes, will be generated (Figure 2B). Remarkably, transfection of the genomic cDNA instead of the antigenomic cDNA greatly impairs or completely abolishes rescue efficiency^2,28-30. Even when antigenomic cDNA is transfected, the efficiency of the encapsidation of the recombinant RNA into RNPs in transfected cells is probably very low. Because of this, rescue protocols for NDV often include different steps for the amplification of the few viral particles released from the originally transfected cells by coculturing them with permissive cells and/or by the infection of embryonated eggs.

Prior to the rescue, the cDNA can be manipulated by standard cloning procedures in order to generate the desired modifications. While specific mutations of the different gene products and regulatory sequences of the virus can be straightforwardly achieved this way, many of the published work involving recombinant NDV has required the addition of a new transcriptional unit into the NDV genome. Like other members of the paramyxovirus family, the NDV genome encodes eight different proteins into six transcriptional units, which are differentially expressed depending on their location respect to the 3' end in a decreasing gradient critical for the viral life cycle¹. Because of this, the location of the new transcriptional unit within the genome must be carefully chosen to achieve a balance between expression of the transgene and impairment of viral replication. Insertion between P and M genes has been used the most, though other sites have also been tested^13,31.

Whatever the insert, the cloning into NDV cDNA needs to follow some rules to generate a rescuable construct: (i) any new gene to be included into the NDV genome has to be under control of the appropriate signals for the viral RNA-dependent-RNA polymerase. These sequences must be added upstream of the new open reading frame (ORF) so the polymerase can recognize the end of the previous gene (GE) and the beginning of the new transgene (GS), spaced by a single nucleotide intergenic sequence (IG). Addition of a valid Kozak (K) sequence to improve eukaryotic ribosomal translation is also recommended for better foreign protein expression³²; (ii) efficient replication of NDV, as for most members of the Paramyxoviridae family, is dependent on the genome length being multiple of six³³; therefore, any insertion into the NDV has to follow this "rule of six". If necessary, required additional nucleotides can be added downstream the new ORF; and (iii) the sequence of the transgene should be checked to find possible GE and GS like sequences which could impair rescue efficiency, transgene expression and/or virus viability. If present, these sequences must be removed by silent mutagenesis. The generation of recombinant full length cDNA following aforementioned rules is the first step in order to efficiently produce a genetically modified NDV as detailed here.

In the system all DNA constructs are under control of the T7 RNA polymerase promoter (Figure 3). This cytoplasmic polymerase is provided in trans by coinfection with a recombinant modified vaccinia Ankara virus (MVA-T7)³⁴. Figure 3A show the pNDV-B1 plasmid, which encodes the full-length antigenomic cDNA⁵. Figure 3B shows pTM1 plasmids encoding NP, P and L ORFs. Plasmids pCITE-GFP, which encodes, under the T7 promoter, the Green Fluorescent Protein (GFP), and pCAGGs GFP¹⁸, which encodes the same ORF under the chicken beta actin promoter ³⁵, are used as controls. In this protocol we show the procedure to rescue recombinant NDV from the cDNA of the lentogenic NDV strain Hitchner B1⁵ (Figure 4).

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 Virus Expressing the Bacteriophage T7 RNA Polymerase (MVA-T7) (Figure 4A, Day 2)

1. Warm up PBS 1x/BA/PS and media at 37 °C.
2. Wash cells, twice, with 1 ml of PBS 1x/BA/PS.
3. Infect the cultured mammalian cells with the MVA-T7 virus at a multiplicity of infection (moi) of 1 pfu/cell in a final volume of 200 μl in PBS/BA/PS for 1 hr at room temperature.
4. During viral infection incubation, prepare the transfection mix as described in 3.

3. Transfection of Mammalian Cells (Figure 4A, Day 2)

1. Preparation of Lipofectamine: Mix 1-2 μg of LPF2000 with 250 μl of OptiMEM per rescue attempt and incubate for 5 min at room temperature.
2. Preparation of DNA: Make a plasmid cocktail for each rescue in 50 μl of OptiMEM. Add 0.4 μg of pTM1-NP, 0.2 μg of pTM1-P, and 0.2 μg of pTM1-L per tube. Add 1 μg of the full-length cDNA clone of NDV to be rescued to each tube. Also prepare two control transfections, one with 2 μg of pCAGGs-GFP (to check transfection efficiency) and the other with 2 μg of pCITE-GFP (to check T7 polymerase driven expression by the MVA-T7).
3. After the LPF2000-OptiMEM solution has been preincubated for 5 min (step 3.1), add 250 μl of the solution to the DNA tubes (step 3.2) and incubate for 20-30 min at room temperature.
4. After incubation with MVA-T7, remove the virus inoculum by aspiration and replace with the DNA-LPF2000 transfection mix (step 3.3).
5. Add 1 ml of DMEM 10% FBS/PS to each dish.
6. Incubate the infected/transfected cells for 6-8 hr (or overnight) at 37 °C, 5% CO₂ and then replace the transfection media with 1.5 ml of fresh DMEM 10% FBS/PS.
7. At 24 hr post-transfection, control wells can be observed under a fluorescence microscope to assess transfection efficiency (pCAGGs-GFP) and T7 polymerase activity (pCITE-GFP) (Figure 5A). Proceed with the co-culture of the infected/transfected cells with avian fibroblasts as described below.

4. Co-culture of Mammalian Cells with Avian Cells (Figure 4A, Day 3)

Usually, a confluent 100 mm tissue culture dish of chicken (CEFs) or duck (DEFs) embryo fibroblasts is used per two transfected wells. Be sure to prepare, in advance, enough 100 mm tissue culture dishes of avian cells per all rescue attempts. For efficient rescue of the virus, DMEM 10% FBS/PS media is supplemented with 5% of allantoic fluid and 30 mM MgCl₂. At this point, 24 hr p.i., mammalian cells may start showing cytopathic effect (CPE) due to MVA-T7 infection, asevident in Figure 5A.

1. Warm up PBS 1x and media to 37 °C.
2. Wash mammalian cells, 2x, with 1 ml of PBS 1x.
3. Trypsinize mammalian cells with 0.2 ml of EDTA-trypsin until they detached. Resuspend the cells in 1 ml of DMEM 10% FBS/PS, 5% allantoic fluid, 30 mM MgCl₂ and transfer to a 100 mm tissue culture dish. Add 3 ml of same media.
4. Wash avian cells, twice, with 4 ml of PBS 1x.
5. Trypsinize avian cells with 1 ml of EDTA-trypsin and incubate for 1-2 min at 37 °C until cells detach. Resuspend the cells adding 8 ml of DMEM 10% FBS/PS, 5% allantoic fluid, 30 mM MgCl₂ and add 4 ml of the trypsinized cells to the mammalian cells in the co-cultured 100 mm tissue culture dish for a total volume of 8 ml (4 ml mammalian, 4 ml avian cells).
6. Gently shake by hand the co-cultured cells in the 100 mm dish in order to have a uniform distribution and then place them in the incubator at 37 °C for 3-4 days. The criteria for infecting eggs 3 or 4 days after co-culture of mammalian and avian cells depends on how much cytopathic effect (cell rounding, death, detachment from the surface, etc.) is observed in the MVA-T7 viral infection.

5. Infection of Chicken Embryonated Eggs (Figure 4A, Days 6-7)

1. After 3-4 days of co-culture of mammalian and avian cells, remove 1 ml of the tissue culture supernatant and add to an Eppendorf tube.
2. Centrifuge the tissue culture supernatant for 1 min at 12,000 rpm in a bench top centrifuge to remove cellular debris.
3. Candle the eggs using a light candling box to see the interface between the air sac and the allantoic cavity. Make a mark on the interface border avoiding blood vessel localization. Spray 70% ethanol over the eggs to establish sterile conditions. Gently make a hole in the eggshell on the marked spot and inoculate 500 μl of the supernatant using an insulin (1 ml) syringe, aiming the needle vertically and directly into the allantoic cavity. (Figure 4B).
4. Seal the nick in the eggshell with melted wax or paraffin.
5. Incubate the eggs for 2-3 days at 37 °C.

6. Harvest of Allantoic Fluid from Infected Chicken Embryonated Eggs (Figure 4A, Days 8-10)

1. Incubate the infected eggs for 2-4 hr or overnight at 4 °C in order to kill the chicken embryo and coagulate the blood.
2. Spray the eggs with 70% ethanol (to maintain sterile conditions).
3. Carefully tap the apical section of the egg, over the air cavity, with a spoon. Once the eggshell is cracked, remove the fragments with forceps and fully expose the allantoic membrane.
4. Expose the allantoic cavity by excising the allantoic membrane with forceps and scissors, avoiding damage to the blood vessels and the yolk.
5. Carefully push down the embryo with a spatula and collect the upflowing allantoic fluid (8-12 ml per egg) with a 10 ml pipette. Avoid damaging the yolk membrane. Transfer the allantoic fluid to a 15 ml centrifuge tube on ice (use one tube per egg).
6. Clarify the allantoic fluid by centrifuging for 5 min at 1,500 rpm. Transfer the supernatant to a fresh tube without disturbing the pellet.
7. Store samples at 4 °C for up to 1 week, until checking them for presence of NDV by hemagglutination assay.

7. Hemagglutination (HA) Assay

The presence of virus in the allantoic fluid from infected eggs can be determined macroscopically by their ability to hemagglutinate turkey red blood cells (RBC). In the case of NDV, approximately 10⁶ plaque forming units (pfu) per ml are required to give a positive signal in the HA assay. HA assays are carried out in V-bottom 96-well plates. Negative (PBS 1x, uninfected allantoic fluid) as well as positive (allantoic fluid from any NDV virus) control samples should always be included in any HA assay to validate it. To perform an HA assay:

1. Pipette 50 μl of PBS 1x per well in a V-bottom 96-well plate.
2. Pipette 50 μl of the allantoic fluid samples into the wells on the first column of the plate. Perform 2-fold serial dilutions through the rest of the plate and discard the last, extra 50 μl from wells in the last column.
3. Dispense 50 μl of 0.5% turkey RBC in PBS 1x per well. Gently shake the plate by tapping.
4. Incubate the plate at 4 °C (or ice) for 30-45 min or until a clear pellet is formed in the negative control wells.

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.