Development of a novel Newcastle disease virus (NDV) neutralization test based on recombinant NDV expressing enhanced green fluorescent protein
© The Author(s). 2017
Received: 14 September 2017
Accepted: 15 November 2017
Published: 23 November 2017
Newcastle disease is one of the most important infectious diseases of poultry, caused by Newcastle disease virus (NDV). This virus is distributed worldwide and it can cause severe economic losses in the poultry industry due to recurring outbreaks in vaccinated and unvaccinated flocks. Protection against NDV in chickens has been associated with development of humoral response. Although hemagglutination inhibition (HI) assay and ELISA do not corroborate the presence of neutralizing antibodies (nAbs); they are used to measure protection and immune response against NDV.
In this study, we established a system to recover a recombinant NDV (rLS1) from a cloned cDNA, which is able to accept exogenous genes in desired positions. An enhanced green fluorescent protein (eGFP) gene was engineered in the first position of the NDV genome and we generated a recombinant NDV carrying eGFP. This NDV- eGFP reporter virus was used to develop an eGFP-based neutralization test (eGFP-NT), in which nAbs titers were expressed as the reciprocal of the highest dilution that expressed the eGFP.
The eGFP-NT gave conclusive results in 24 h without using any additional staining procedure. A total of 57 serum samples were assayed by conventional neutralization (NT) and eGFP-NT. Additionally, HI and a commercial ELISA kit were evaluated with the same set of samples. Although HI (R 2 = 0.816) and ELISA (R 2 = 0.791) showed substantial correlation with conventional NT, eGFP-NT showed higher correlation (R 2 = 0.994), indicating that eGFP-NT is more accurate method to quantify nAbs.
Overall, the neutralization test developed here is a simple, rapid and reliable method for quantitation of NDV specific nAbs. It is suitable for vaccine studies and diagnostics.
Newcastle disease virus (NDV) is the causal agent of a highly contagious and fatal disease that affects poultry and other avian species worldwide . Virulent strains of NDV are capable of causing high mortality (up to 100%) in non-vaccinated chickens . NDV is a member of the genus Avulavirus of the family Paramyxoviridae in the order of Mononegavirals . This virus has a nonsegmented single-stranded negative-sense RNA genome, which contains a 3′- leader and a 5′- trailer sequences, essential for virus transcription and replication, and follows the rule of six . NDV possess six structural genes: Nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN) and large polymerase (L) . From these proteins, N, P and L proteins form the Ribonucleoprotein (RNP) complex, which is responsible for viral transcription and replication . HN and F are anchored in the viral envelope as surface glycoproteins: HN is responsible for the attachment of the virus to the host cell receptor, and F mediates fusion of the viral envelope with the host cell membrane . The F protein is proteolytically cleaved to F1 and F2 for fusion activity and the presence of a polybasic motif in the cleavage site is a major determinant of virulence [6, 7]. Both HN and F proteins are capable of eliciting neutralizing antibodies (nAbs) [8–12].
Humoral immunity plays an essential role in the protection against NDV infection. Chickens with high antibody titers are usually protected. For example, young chicks with high maternal antibody titers are protected against a challenge with a virulent strain during the first few days . Protection against the virus has been described for chickens passively immunized with egg yolk or antiserum from hyperimmunized birds against the whole virion. Monoclonal antibodies against HN and F proteins are able to neutralize the virus, both in vitro and in vivo [13–15]. Although, antibodies against F and HN have a synergistic potential . Recently, higher and specific levels of antibodies were not only related with protection against mortality, but also with reduction of viral replication and secretion . Hence, measuring the neutralizing antibodies (nAbs) against NDV is highly essential to evaluate the efficacy of a vaccine.
Usually, hemagglutination inhibition (HI) assay and Enzyme-Linked ImmunoSorbent Assay (ELISA) are used to measure NDV-specific antibodies but not necessarily nAbs against NDV. Conventional neutralization test (NT) is laborious, time-consuming and may have operator bias. Therefore, a rapid, high-throughput and reliable NT assay is necessary for evaluation of NDV nAbs. In recent years, few researchers have shown that genetically engineered viruses expressing the green fluorescent protein (GFP) or the enhanced GFP (eGFP) can be used for rapid determination of virus neutralizing antibody titers or antiviral activities [17–21]. The eGFP expressed by these viruses allows direct visualization of the infection under a fluorescent microscope or its automatization by using a fluorescence reader plate. These characteristics make it a suitable method to overcome the drawbacks of a conventional NT.
In this report, we describe the generation of a genetically engineered NDV expressing the eGFP from cDNA, and development of an eGFP-based NT (eGFP-NT) for rapid detection of NDV nAbs. Our results show that this method is fairly accurate as a conventional NT method but a better alternative in terms of being cost-effective and efficient.
Two cell lines were used in this study, DF-1 (derived from chicken fibroblasts) and Vero (monkey kidney cells), which were purchased from ATCC (Manassas, VA, USA). Both cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM) F12 (HyClone) supplemented with 5% heat-inactivated fetal bovine serum (FBS), 2.5% chicken serum (ChkS) (Sigma–Aldrich), 100 U/mL of penicillin and 100 μg/mL of streptomycin at 37 °C in an atmosphere of 5% CO2.
Construction of a full-length clone of NDV
Nucleotide differences between the reference NDV and the assembled full-length clone, pFLC-LS1
Creation of BssHII
Creation of NruI
Creation of MluI
Creation of SnaBI
per se mutation
per se mutation
per se mutation
per se mutation
Destruction of BbvCI
Construction of supporting plasmids
Oligonucleotides used for cloning of the supporting plasmids, construction of pFLC-LS1-1eGFP, and confirmation of virus recovery
Sequences (5′ → 3′)a,b
Supporting plasmid primers
M + 4100 d
Construction of pFLC-LS1-1eGFP plasmid
Transfection and virus recovery
High-quality plasmid DNA was obtained from the pFLC-LS1, pFLC-LS1-1eGFP and three supporting plasmids using the Plasmid Midi Kit (Qiagen). Vero cells were grown to 80% confluency in a 12-well plate and then co-transfected with 1 μg of pCI-N, 0.5 μg of pCI-P, 0.2 μg of pCI-L and 2 μg of pFLC-LS1 or pFLC-LS1-1eGFP, using the Lipofectamine™ LTX & plus Reagent (Invitrogen, USA) according to manufacturer’s instructions. Briefly, plasmids were diluted in a 400 μl of Opti-MEM medium (Invitrogen, USA). Next, Lipofectamine was added and incubated for 15 min at room temperature. Vero cells were washed with Dulbecco’s Phosphate Buffered Saline (DPBS) and then the plasmid-Lipofectamine mixture was added to the monolayer. After 4 h of incubation at 37 °C, transfected cells were washed and then maintained in DMEM containing 5% FBS at 37 °C and 5% CO2. Next day, allantoic fluid (AF) was added to a final concentration of 5% and cells were incubated for 4 more days. Cell supernatant was harvested, pelleted at high speed and inoculated into 8-days old SPF chicken embryonated eggs and incubated for 4 days. AFs were harvested, clarified, and aliquoted and stored at −80 °C. Recovery of the viruses was first confirmed by hemagglutination assay (HA). For parental virus (rLS1), the presence of NDV-specific protein was evaluated by immunofluorescence and confirmation of genetic markers was performed by RT-PCR and restriction enzyme digestions. To confirm the recovery of rLS1-1eGFP virus, infected DF-1 cells were observed under a fluorescence microscope.
Immunofluorescence of rLS1
DF1 cells infected with the rLS1 virus at a multiplicity of infection (MOI) of 0.01 for 48 h. After discarding the supernatant, the cells were washed with DPBS 3 times (as all washing steps in this section) and fixed with 4% paraformaldehyde in DPBS for 15 min at room temperature. Fixed cells were washed, permeabilized with 1% SDS in DPBS for 15 min at room temperature and washed again. Permeabilized cells were blocked with 5% Bovine Serum Albumin (BSA) (Sigma-Aldrich) in DPBS for 30 min at room temperature. Cells were incubated for 2 h with a monoclonal antibody against the NDV ribonucleoprotein (RNP) (cat. n° ab138719, Abcam, USA) at a final concentration of 3.85 μg/mL in a solution of 5% BSA in DPBS. After washing, the cells were incubated for 1 h with a goat polyclonal antibody anti-mouse IgG labeled with Alexa Fluor 594 (cat n° ab150116, Abcam) at a final concentration of 1 μg/mL in a solution of 5% BSA in DPBS, then washed. Nuclei were stained with DAPI for 5 min. Cells were examined under the Observer.A1 fluorescent microscope (Carl Zeiss, Germany). The fluorescent signal images were taken at 400X magnification with the AxioCam MRc5 camera (Carl Zeiss, Germany).
RT-PCR and confirmation of genetic markers
The identity of genetic markers in recombinant NDVs was confirmed after RT-PCR amplification from genomic RNA and sequencing of the DNA fragments. Two genetic markers evaluated were; creation of the BssHII site in the M gene and destruction of the BbvCI site in the L gene (see Table 1). Briefly, viral RNA was extracted from AFs stocks using the QIAamp Viral RNA mini kit (Qiagen) and cDNA was generated using the ProtoScript® II First Strand cDNA Synthesis Kit (New England Biolabs Inc). The PCR reactions were performed with primers M + 4100  and NDV4394_R to amplify the region containing the BssHII site, and primers NDV14035_F and NDV15003_R for BbvCI site that was destroyed (see Table 2). Restriction analysis of the PCR products was carried out on a 2% agarose gel.
DF-1 cells were seeded into 12-well plates at density 1.5 × 105 cells in 1 mL of DMEM F12 per well, a day before the assay. Next day, serial dilutions of 1:10 were made by mixing 25 μL of virus with 225 μL of serum free DMEM F12. After washing DF-1 cells monolayer with DPBS, dilutions (0.2 mL/well) were added into wells and incubated for 1 h at 37 °C in an atmosphere of 5% CO2 for viral absorption. Later, the inoculum was removed and each well was covered with 1 mL of an overlay medium consisting of DMEM supplemented with 0.5% agarose, 5% AF and 30 mM MgCl2 . The overlay medium was allowed to solidify by placing the plates on a level surface at room temperature for 15 min, the plates were then incubated for 5 days at 37 °C and 5% CO2. The plates were fixed and stained with a mixture of 0.2% crystal violet (Sigma-Aldrich) and 3.2% paraformaldehyde overnight at room temperature . The plates were rinsed with tap water, dried, viewed and photographed for EliSpot (EliSpot Reader versión 7.0). The NDV titers were reported as plaque-forming units per milliliter (PFU/mL).
Virus growth curves in DF-1 cells
Monolayer cultures of DF-1 cells were seeded at 50–60% confluence in 12-well plates and infected with rLS1 or rLS1- eGFP viruses at a (MOI) of 0.05. Cells were cultured with DMEM containing 1% FBS and 5% AF with 5% CO2 at 37 °C. Supernatants were collected 12, 24, 36, 48, 60 and 72 h post-infection (h.p.i.). Collected supernatants were quantified in DF-1 cells by plaque assay as described above.
Serums samples obtained from 57 chickens were used for the experiments. Thirty seven serum samples were collected from field vaccinated chickens from different farms. Six commercially available chicken sera (Charles River Laboratories), corresponding to sera anti-Marek’s disease virus (MDV), anti-Avian adenovirus type-1, anti-Infectious Laryngotracheitis virus (ILTV), anti-Infectious bronchitis virus (IBV), anti-NDV, another anti-NDV serum (cat. n° ab34402, Abcam), and a serum from a SPF chicken was included as negative control. The remaining 13 serum samples were obtained from SPF chickens inoculated with one dose of LaSota vaccine at 1 day of age and their sera taken at the 4th week. All serum samples were heat inactivated (56 °C for 30 min) and stored at −20 °C . Detailed information about the serum samples is listed in Additional file 1: Table S1.
Conventional NT and eGFP-NT were carried out to quantify nAbs in chicken sera. For both assays, serum samples were titrated in duplicate, using 96-well flat bottom nonpyrogenic polystyrene culture plates (Corning, NY, USA). A day before the assay, 1 × 104 DF-1 cells per well were seeded in 0.1 mL of DMEM F12 supplemented with 5% FBS. The serum samples were serially diluted by 2-fold (starting from 1:2) with DMEM F12 serum free medium, supplemented with FA and FBS at final concentrations of 5% and 1%, respectively. Serum dilutions were mixed with 100 PFU of virus (rLS1 or rLS1-1eGFP, respectively). Each dilution was evaluated in duplicate. For conventional NT, DF-1 cells were infected with mixtures of virus-serum. After 4 days of incubation at 37 °C in 5% CO2, cells were washed with DPBS. Then cells were fixed and stained with a mixture of 0.2% crystal violet and 3.2% paraformaldehyde for 15 min at room temperature. NDV nAb titers were determined as the reciprocal of the highest dilutions that both replicates presented a clear CPE.
For eGFP-NT, once DF-1 cells were infected with mixtures of virus-serum, cells were incubated for 48 h and observed under a fluorescence microscope and NDV nAb titers were determined as the reciprocal of the highest dilutions that did not express the eGFP in any of the duplicates. Wells with one or more fluorescent foci were considered positive .
Hemagglutination inhibition (HI) test
HI was performed according to the OIE terrestrial manual . Briefly, 25 μL of PBS per well were dispensed in clear 96-well V-bottom plates, then 25 μL of serum was placed on the first well, 2-fold dilutions of 25 μL of the serum suspension were made across the entire plate. Then, 25 μL of diluted virus containing 24 hemagglutination units (HAU) of rLS1 virus was added to each well and incubated for 30 min at room temperature. Next, 25 μL of chicken red blood cells (RBCs) at 1% were added to each well and incubated for 40 min at room temperature. The titration was determined as the highest dilution of serum causing complete HI.
ELISAs against NDV (IDEXX laboratories, USA) were performed on all serum samples at room temperature, according to manufacturer’s instructions. Briefly, 100 μl of each serum sample (diluted 1:500 in DPBS), and 100 μl of the negative and positive control samples were dispensed into duplicate wells and incubated for 30 min. The plates were then washed thrice and incubated with 100 μl of conjugate per well for 30 min. Wells were washed thrice and then 100 μl per well of substrate solution was added, incubated in the dark for another 30 min, after which 100 μl stop solution was immediately added. The plates were read using an Epoch 2 microplate reader (Biotek, USA) at 450 nm. Data obtained were analyzed and sample to positive ratio (S/P) calculated.
The t-student test was performed to compare the mean titer of each time of the growth kinetics curve. Linear regressions were performed for the analysis of correlation between NT and eGFP-NT, HI and ELISA titers. All statistical analysis were performed in GraphPad Prism 6.01 (GraphPad Software Inc., San Diego, CA).
Construction and characterization of the recombinant rLS1 NDV
Construction and characterization of the rLS1-1eGFP reporter NDV
Comparison of growth kinetics of the recovered viruses was performed in DF-1 cells at an MOI of 0.05. Both viruses exhibited similar growth characteristics. However, rLS1-1eGFP showed significantly lower titers than rLS1 (p < 0.01) at 24 and 36 h.p.i. (Fig. 4b). The highest titers achieved in cell culture for both viruses were approximately 106 PFU/ml (Fig. 4b), whereas the titers in AF collected from SPF embryonated eggs were at least 108 PFU/ml (data not shown).
The correlation between eGFP expression level and the efficiency of viral replication was assessed by infecting DF-1 cells at different MOIs with rLS1-eGFP, and recording these cells every 12 h. The intensity and number of cells expressing the eGFP were increasing in a time- and dose-dependent manner (Fig. 4c). The eGFP expression can be clearly visualized as early as 24 h.p.i., although it can be detected with low expression at 12 h.p.i. These results indicates that eGFP expression can be used to monitor viral replication.
Strong correlation between conventional NT and the eGFP-NT
Conventional- and eGFP-NT were first evaluated by using diluted reference chicken serum samples. Reference serum samples, positive to other pathogens (IBV, MDV, Avian adenovirus type 1 and ILTV) and from a SPF chicken, were used to evaluate non-specific neutralization. These sera tested negative, as expected, for both neutralization methods (See Additional file 1: Table S1). Fluorescence in the eGFP-NT was visible as early as 18 h of incubation, and it was clearly visible at 24 h.
HI and ELISA have strong correlations with conventional NT but present greater dispersion than eGFP-NT
Correlations between nAbs titers and the antibody titers measured by HI and ELISA were evaluated. In Fig. 5b, the relation between log2 HI and log2 NT is shown. A strong correlation (R 2 = 0.816) was observed between NT and HI titers. In addition, several samples that tested positive to the HI assay, with titers corresponding to dilutions of up to 1:8, were negative for neutralization. These results are consistent with the fact that the equation of the line (Y = 0.816X + 1.238) shows that when NT is 0, in average, HI titers would be positive and greater than a titer corresponding to a dilution of 1:2. It is worth mentioning that serum samples positive to HI and negative to the NT assay came from vaccinated chickens (see Additional file 1: Table S1), suggesting that either nAbs are not circulating at that time because plasma cells become memory B cells or specific antibodies against NDV exist, but these are not nAbs.
Antibody titers measured by ELISA (S/P) also have a strong correlation (R 2 = 0.791) with the log2 NT titers (Fig. 5c). Both HI and ELISA showed greater dispersion to the conventional NT than the eGFP-NT. These results indicate that HI and ELISA are not as reliable as eGFP-NT, showing that this method can close the gap between a rapid and accurate measurement of nAbs against NDV.
NDV is one of the major threats to the poultry industry worldwide and new genotypes and sub-genotypes are discovered every year . In South America, the virus is currently circulating [38–41], and there is an urgent need to improve vaccines as well as biosafety and surveillance. The relevance of nAbs in protection and against viral shedding has been well established [8, 12, 16]. Thus, NDV vaccines should be capable of eliciting high nAb titers. A rapid and easy-to-perform method to measure nAbs will help to evaluate new vaccine candidates. In this context, we developed a new vector (rLS1), which can accept inserts in all intergenic regions. Later, the eGFP gene was incorporated before the N gene into the rLS1 virus genome, generating the rLS1-1eGFP. We chose that position to produce high levels of eGFP protein . This virus was used to develop the eGFP-NT to measure nAbs.
In our hands, conventional NT took 4 days to distinguish infected wells from non-infected ones, which were difficult to read from stained neutralization plate wells. In contrast, results from eGFP-NT can be obtained within a day. We set 24 h.p.i. as a conservative time to observe infected cells to measure nAb titers because at this time eGFP expression is very obvious in positive wells. Nevertheless, we obtained the same nAb titers when measured at 18 h or later (data not shown). For conventional NT, the difficulty to clearly differentiate infected from non-infected wells in limiting dilutions can lead to an operator bias. In contrast, eGFP-NT is a reliable tool, in which fluorescence can be easily detected, minimizing operator bias.
Conventional NT exhibited stronger correlation with the eGFP-NT (R 2 = 0.994) than ELISA (R 2 = 0.791) or HI (R 2 = 0.816). Although our data suggest good correlations between nAbs and HI or ELISA, there is window of uncertainty at low titers in both cases. Based on our regressions, HI assays overestimate and ELISA underestimate NT titers. Furthermore, these equations can be heavily affected based on the origin of the immunogen responsible to elicit humoral response. Several groups have shown that antibody titers measured by ELISA or HI do not necessarily correspond with NDV nAb titers [8, 12, 13]. Our results also show that HI tends to produce positive results in some samples with negative nAb titers, and both HI and ELISA have a big dispersion with respect to NT. In contrast, eGFP-NT has evident advantage over HI or ELISA because it clearly shows the presence of nAbs induced after vaccination. It is highly probable that correlations will dramatically change when subunit or virus-vectored NDV vaccines are used for immunization because only some epitopes will be displayed, but they will not necessary induce the production of antibodies detected by HI or ELISA. For example, avian paramyxovirus type 3 vectors carrying either F or HN proteins or the combination of both vectors were capable of eliciting similar nAb titers against LaSota-NDV control, but failed to elicit ELISA titers . In other study, virus-like particles (VLP) based vaccine of NDV F protein and influenza M1 protein has been tested in SPF chickens, which gives low levels of NDV ELISA titers but still shows that birds were fully protected . Similar results have been observed in fowl pox vectored NDV vaccine  and in a baculovirus expressing the F protein .
Recently, an NDV-pseudotyped HIV was engineered to express both F and HN proteins from NDV along with a luciferase reporter protein, and this virus was used for neutralization assays . In our case, we have designed the NDV vector in such a way that it can accept inserts in almost all intergenic regions and one can exchange F and HN genes from various donors. The pFLC-LS1-1eGFP plasmid, containing the reporter eGFP gene in the NDV backbone, has unique restriction sites flanking the F (BssHII and MluI) and HN (MluI and SnaBI) genes, which can be used to easily replace the genes of other genotype. Additionally, the use of a reporter NDV expressing the eGFP has some advantages when compared to pseudotyped lentivirus vector that was used for neutralization assays: 1) Production of rLS1-1eGFP virus is technically easy, as the virus grows to high titers in both cell culture and embryonated eggs, allowing us to prepare large viral stocks. In contrast, lentiviruses require a new transfection procedure each time to generate the reporter virus . 2) As mentioned earlier, eGFP-NT is cheaper, and does not require additional reagents. In contrast, lentiviruses expressing the luciferase enzyme require luciferase reporter kits and a luminometer to be quantified. 3) rLS1-1eGFP can be used for high throughput assays by measuring eGFP positive cells with a fluorescence reader directly in plates without any staining [18, 20, 21]. 4) Finally, rLS1-1eGFP vector can be used to express foreign F and HN in its natural context instead of using a surrogate system that may not reflect the same entry mechanism and therefore infectious efficacy.
We generated a recombinant NDV harboring the eGFP from cloned cDNA, and developed an eGFP-based neutralization test (eGFP-NT) for rapid detection and quantification of nAbs against NDV. This novel test is simple, inexpensive, and accurate, which makes it suitable to test new NDV vaccine candidates. The eGFP-NT can be implemented in laboratories with basic cell culture equipment and a florescent microscope. So far, it is the quickest method to evaluate NDV nAbs, with a higher correlation to the conventional neutralization test.
The authors would like to thank Angela Montalvan and Edison Huaccachi for their excellent technical assistance.
This study was partially funded by the Peruvian Science and Technology Program (FINCyT) contract n° ITAI-2-P-050-009-15.
Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information files.
AC, RI-L, MF-D and VV contributed to conception and designed the research. AC, RI-L and KC acquired the data of the study. AC and RI-L analyzed the data, and all authors interpreted them. AC, RI-L and VV drafted the paper, and all authors revised it critically, read and approved the final manuscript.
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