Development and evaluation of a chicken embryo fibroblast cell culture based live attenuated Indian strain duck plague vaccine

Abstract

Duck plague (DP) is an acute, contagious and fatal disease, caused by duck enteritis virus (DEV), with worldwide distribution causing several outbreaks and posing severe economic losses. The present study was carried out with a goal of development of a live attenuated cell culture based DP vaccine using an Indian strain of DEV and evaluation of its safety, efficacy along with complete genome analysis. The live attenuated DP vaccine (DPvac/IVRI-19) was developed by serial propagation of a virulent isolate of DEV (DEV/India/IVRI-2016) in the chicken embryo fibroblast (CEF) primary cell culture. Adaptation of DEV in CEF cell culture was indicated by more rapid appearance of cytopathic effects (CPE) and gradual increase of virus titre, which reached up to 10^7.5 TCID₅₀/mL after 41 passages. The safety, immunogenicity and efficacy of the vaccine were determined by immunization trials in ducklings. The DPvac/IVRI-19 was found to be avirulent and completely safe in the ducklings. Further, the vaccine induced both humoral and cell mediated immune responses and afforded 100% protection against the virulent DEV challenge. A comparison of the whole genome of DPvac/IVRI-19 (MZ911871) and DEV/India/IVRI-2016 (MZ824102) revealed significant number of mutations, which might be associated with viral attenuation. Phylogenetic tree of DEV/India/IVRI-2016 revealed its evolutionary relationship with other DEV isolates, but it formed a separate cluster with certain unique mutations. Thus, with the proven safety and 100% efficacy, the DPvac/IVRI-19 is suitable for large scale production with precisely pure form of vaccine and has potential utility at national and global levels.

Keywords:

• Duck enteritis virus
• duck plague
• chicken embryo fibroblast
• cell culture attenuated vaccine
• safety
• efficacy
• complete genome analysis

1. Introduction

Duck viral enteritis (DVE) or Duck plague (DP) is an acute, sometimes chronic contagious and fatal virus infection affecting the Anatidae group of birds including ducks, geese and swans. Among various diseases affecting the duck population, DP is the most significant viral disease reported worldwide, causing huge economic losses due to high morbidity and mortality (Campagnolo et al. 2001; Dhama et al. 2017). The DEV or Anatid herpesvirus-1 belongs to the genus Mardivirus in the subfamily Alphaherpesvirinae of the family Herpesviridae (Sandhu and Metwally 2008). It is a double-stranded, enveloped DNA virus with icosahedral symmetry and the genome is 158,091 bp in length, with 76 coding genes and a GC content of 39.9% (Dandapat et al. 2022). From 1923 until the present, there have been several outbreaks of DP throughout the world (Baudet 1923; Pechan et al. 1985; Kaleta et al. 2007; Islam et al. 2021; Liang et al. 2022). In India, the disease prevalence has also been increasing with the timeline (Mukerji et al. 1965; Rajan et al. 1980; Dhama et al. 2017; Neher et al. 2019; Pazhanivel et al. 2019). The disease was noticed in all age group of birds with an incubation period ranging from 3 to 7 days. Clinical signs mainly include photophobia, ataxia, severe enteritis, watery diarrhoea, and sick birds keeping their stance upright by using their wings for support (OIE Terrestrial Manual 2018). Migratory waterfowls play a crucial role in disease transmission, which carry pathogens and infect the susceptible flocks.

The main strategy to prevent and mitigate the spread of DVE is only through vaccination and building immunity against the virus. Jansen et al. (1963) in Netherland first developed an attenuated chicken embryo-adapted vaccine through multiple passages in chicken embryos, following that, the vaccine was used globally. In Vietnam, a Chinese strain originally adapted to embryonated duck eggs underwent 15 serial passages in embryonated chicken eggs and 12 passages in chicken embryo fibroblast (CEF) cell cultures to develop the vaccine, which was shown to be safe and efficacious against the Vietnam challenge virulent strains (Dinh et al. 2004). Hossain et al. (2005) prepared an experimental live attenuated vaccine using a local virulent isolate of duck plague virus (DPV) in Bangladesh by initial 5 passages in the embryonated duck eggs followed by 16 passages in embryonated chicken eggs (ECE) and reported that the experimentally prepared vaccine induced higher immune response than the conventional vaccine. A virulent Chinese strain was serially propagated up to 80 passages in a 9-days-old ECE, that has passed safety and potency testing and is used in commercial vaccine production in China (Qi et al. 2009). In another report, the Chinese challenge virus was serially passaged 20 times in CEF cell culture and subsequently 85 times in chick embryos, resulting in 22 nucleotide substitutions and single amino acid change in open reading frames (Yang et al. 2017).

Currently, a chicken embryo-adapted live attenuated DEV vaccine is being commercially produced in India using the Holland or Jansen strain, which was imported in 1979. However, considering the large population of domestic and wild ducks in the country, the production and supply of the vaccine remain insufficient. Partially purified egg-adapted vaccine preparations are in a crude form that contains extraneous substances like chicken embryo tissues and other egg-associated proteins, which may unnecessarily burden the immune system of the vaccinated birds. Further, it involves a cumbersome method of propagating the vaccine virus in ECE, which is not convenient for large-scale production and difficult to ascertain the titre of vaccine from batch to batch as the virus titration again involves a large number of ECE. A study on comparative efficacy revealed that vaccine prepared from the locally prevailing strain produces a better humoral and cell-mediated immune response than the foreign strains (Kulkarni et al. 1998). Few reports of clinical DP diseases in vaccinated flocks further underlines the incomplete protection offered by foreign strains, that may be attributed to the lack of antigenic relatedness of the vaccine with field strains along with inadequate vaccine quality and vaccination practices (Rajan et al. 1980; Rani and Muruganandan 2015). Therefore, a precisely pure form of vaccine using a prevalent local strain of DEV may provide an opportunity to reduce the outbreaks by enhancing herd immunity through a mass vaccination program and should be amenable for industrial-scale production. With this background, the present study was carried out to develop a cell culture-based DP vaccine using an Indian field isolate (DEV/India/IVRI-2016), which was adapted and attenuated through serial propagation in CEF cell culture. Further, the attenuated vaccine strain (DPvac/IVRI-19) was evaluated for safety, immunogenicity and protective efficacy with a comparative genomic analysis of the virulent and attenuated strains.

2. Materials and methods

2.1. Ethical statement

The experimental procedures in ducklings were carried out according to the guidelines and approval of the Institute Animal Ethics Committee (IAEC) and Institute Biosafety Committee (IBSC) of ICAR-Indian Veterinary Research Institute (F.No.26-1/2015-16/JD(R), dated 25.10.2016 and F.No.26-5/2021-22/JD(R)/IAEC Meeting, dated 15.12.2021). The present work does not involve any human participants.

2.2. Duck enteritis virus (DEV)

The DEV/India/IVRI-2016 strain, isolated from the field samples collected during an outbreak in Kerala, India, was characterized as described in our previous study (Panickan et al. 2021) along with the whole genome sequence analysis (NCBI GenBank no. MZ824102) (Dandapat et al. 2022).

2.3. Adaptation and serial propagation of DEV in CEF cell culture

Preceding the adaption of DEV/India/IVRI-2016 in CEF cell culture, the virus was initially propagated in embryonated duck eggs by chorio-allantoic membrane (CAM) route up to 15 passages and subsequently in duck embryo fibroblast (DEF) primary cell culture for another 15 passages (DEF-P15). Finally, the DEF passaged virus was adapted in CEF culture by infection of monolayer (adsorption method 1.5 h) for initial 5 passages and then serially propagated by co-infection with the multiplicity of infection (MOI) rate of 0.01, up to 41 passages (CEF-P41) to achieve attenuation of the virus. The CEF cell monolayers were grown in tissue culture flasks (25 cm²) at 37 °C under 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA), supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and antibiotics. After 24 h of incubation, when the CEF monolayer was almost complete, it was washed thrice with plain DMEM, replaced with DMEM maintenance medium containing 2% FBS and further incubated. Cells were routinely observed for cytopathic effects (CPE) and the culture was harvested, when the optimum characteristic CPE appeared. The flasks were kept frozen at −80 °C and subsequently used after three freeze-thaw cycles.

2.4. Detection of DEV in infected CEF cells by PCR and immunofluorescence test

Presence of the virus (DEV) in the infected cell culture fluid at different passage levels was confirmed by amplification of DNA polymerase gene of DEV using the specific primers in PCR as per OIE Terrestrial Manual (2018). PCR products were resolved on 1% agarose gel electrophoresis.

For localization of viral antigens in CEF culture by indirect immunofluorescence test (IIFT), the CEF cells suspension (10⁶ cells/mL) in DMEM growth medium were dispensed in a 12-well tissue culture plate (1 mL/well) and co-infected with CEF passaged virus at P35 (50 µL/well, titre 10^7.2 TCID₅₀/mL) and incubated for 48 h. CEF cells without virus infection served as negative control. Primary antibody (anti-DEV duck serum) was used at the dilution of 1:100 and anti-duck IgG FITC conjugate (KPL/Sera Care, USA) was added at the dilution of 1:50. Presence of DEV antigen in the infected CEF cell culture was demonstrated under inverted fluorescent microscope (Nikon, Japan).

2.5. Growth kinetics of DEV in CEF cell culture

The growth kinetics of DEV propagated in CEF was determined at the passage level P35. Briefly, the CEF monolayer was infected with DEV at a MOI rate of 0.01 and incubated for 75 min for virus adsorption. The residual virus was removed and replaced with DMEM maintenance medium containing 2% FBS. Cell culture was harvested at different time intervals, i.e. 12, 24, 36, 48, 72 and 96 h post infection (hpi) and stored at −80 °C. Virus titre at different intervals was measured by limiting dilution on CEF cells in 96-well plates and TCID₅₀ per mL was calculated as per previous method (Reed and Muench 1938).

2.6. Sterility testing

The sterility of CEF attenuated vaccine (DPvac/IVRI-19) was confirmed by inoculating on to the bacterial and fungal media (blood agar, nutrient media and Sabouraud Dextrose Agar) and observed for growth for 14 days. The attenuated vaccine virus was also screened for absence of other concomitant avian infectious agents, such as Marek’s disease virus, chicken infectious anaemia virus, egg drop syndrome virus, avian leukosis virus, Newcastle disease virus, infectious laryngotracheitis virus, avian influenza virus, fowl adenovirus and avian mycoplasma by PCR using specific primers.

2.7. Safety testing

Safety testing of the DPvac/IVRI-19 was done as per the method recommended by Indian Pharmacopeia (IP-2018). A group of 10 ducklings (n = 10) of 8 weeks old were inoculated with the vaccine at the dose of 10^4.5 TCID₅₀ (i.e. 10 times of an effective vaccine dose) in 1 mL, subcutaneously and were kept under observation up to 21 days post immunization (dpi) for any adverse reaction or clinical signs.

2.8. Test for reversion to virulence by back passage in ducklings

To establish the stability of attenuated vaccine virus and to investigate any chance of reversion to virulence, a group of 5 ducklings (n = 5) of 8 weeks old were inoculated with 10^3.5 TCID₅₀ vaccine dose. On the 7^th day post inoculation, ducklings were sacrificed and their liver tissue was collected, homogenized and 10% suspension was prepared. The liver homogenate was clarified by low-speed centrifugation at 3,000 rpm for 10 min, and 1 mL of the supernatant was administered subcutaneously to naive ducklings. Similarly, subcutaneous inoculation of ducklings with clarified supernatant of liver tissue homogenate was repeated and was serially passaged for 6 times and after 6^th passage, the birds were kept under observation for a period of 21 days.

2.9. Immunization experiment design

For immunization trial, a total of 75 healthy ducklings of 8-weeks old were divided into five groups (A, B, C, D and E) consisting of 15 birds in each group (n = 15). All the experimental ducklings were tested to be negative for DEV and its antibodies. The birds of group A, B, C and D were immunized subcutaneously with different doses (10^2.5,10^3.5,10^4.5 and 10^5.5 TCID₅₀ in 1 mL, respectively) of CEF passaged (P41) DEV vaccine (DPvac/IVRI-19). Group E birds served as unvaccinated control, which were inoculated with 1 mL of culture medium. Serum samples were collected from all the groups at 7^th, 14^th and 21^st dpi to determine the protective antibody level by virus neutralization test (VNT) and blood samples were also collected at 7^th, 14^th and 21^st dpi for assessment of CMI responses by lymphocyte proliferation assay and enumeration of CD4+ and CD8+ cells in peripheral blood mononuclear cells (PBMCs).

2.10. Determination of virus neutralizing antibody levels

Virus neutralization test (VNT) was performed in 96-well flat bottom tissue culture plates as per the method described by Ning et al. (2022) with minor modifications. Briefly, serum samples collected at 7, 14 and 21 dpi were heat inactivated at 56 °C for 30 min and two-fold serial-dilutions (2¹ to 2⁸) were made. 50 μL of serum from each dilution was pre-incubated with 50 μL of virus suspension in DMEM (containing 200 TCID₅₀) for 90 min at 37 °C. Then, 100 μL of CEF cells (10⁵/well) were added to the serum-virus mixture and incubated for 4-5 days at 37 °C with 5% CO₂. The cell control without virus and the virus control with 200 TCID₅₀ and 20 TCID₅₀/50 μL were also taken in separate set of wells in the same plate. The titre of virus neutralization activity was expressed as the reciprocal of the highest dilution of serum at which there was no evidence of CPE and complete virus neutralization has occurred. The neutralization index was calculated according to the Reed and Muench (1938) method.

2.11. Determination of cellular immune response

2.11.1. Lymphocyte proliferation assay

The cell mediated immune (CMI) response in ducklings of different groups at 7, 14 and 21 dpi was assessed by lymphocyte transformation test (LTT) using MTT colorimetric assay (Mosmann 1983). Briefly, the PBMCs were isolated from the duck blood by density gradient centrifugation using Histopaque-1.077 (Sigma-Aldrich). Cell concentration was adjusted to 2 × 10⁶ cells/mL and dispensed in a 96-well cell culture plate (100 μL/well). Then, RPMI-1640 growth medium (100 μL) with DEV (10⁴ TCID₅₀) was added to the wells in triplicates for assessment of antigen-specific proliferative response (Apinda et al. 2022). Concanavalin A (Con-A, Sigma-Aldrich) @ 20 μg/mL in RPMI-1640 as the positive control and plain growth medium (without antigen or Con-A) as negative control were also taken in triplicate wells. After 48 h incubation at 37 °C with 5% CO₂, 20 μL of 5 mg/mL concentration of MTT (Sigma-Aldrich) dye solution was added to each well and further incubated for 4 h. The formazan crystals formed were dissolved with 100 μL of DMSO per well and optical density (OD) at 570 nm was taken by using a microplate ELISA reader. Lymphocyte proliferative response was expressed as the mean stimulation index (SI), calculated by dividing the mean OD of the stimulated cultures by the mean OD of unstimulated control.

2.11.2. Flow cytometry analysis of CD4+ and CD8+ T-cells

Enumeration of CD4+ and CD8+ T-cell subsets in PBMCs of the vaccinated and control groups was performed on FACS Calibur® instrument (BD Bioscience). 10 μL of anti-duck CD4 monoclonal antibody or anti-duck CD8 monoclonal antibody (AbD Serotec Ltd., UK) was added to the PBMC (5x10⁵cells) in duplicates and incubated at 37 °C for 30 min. Fluorescein isothiocyanate (FITC)-labelled goat anti-mouse IgG (AbD Serotec Ltd., UK) was then added and incubated at 37 °C for 30 min. The cells were washed and resuspended with PBS and then subjected to flow cytometry analysis. Ten thousand cells were analysed in ‘Cell quest’ software of FACS Calibur (BD Bioscience).

2.12. Determination of protective efficacy against challenge infection

For challenge infection in testing potency or efficacy of the vaccine, the virulent field isolate (DEV/India/IVRI-2016) was first revived by inoculating subcutaneously in two healthy ducks, the liver was collected upon their death and 10% (w/v) suspension was prepared with sterile PBS, which was used as the challenge virus. The duckling lethal dose (DLD₅₀) was determined by inoculating different 10-fold serial dilutions (10⁻¹ to 10⁻⁷) of the virulent DEV in different groups of ducklings (n = 3), subcutaneously. All the birds were observed daily up to 14 dpi for the clinical signs and mortality. The DLD₅₀ was calculated as per method of Reed and Muench (1938).

In order to test the potency or efficacy of the vaccine, 10 ducklings of each vaccinated and control group (n = 10) were challenged at 21 dpi with 10² DLD₅₀ and the rest five birds (n = 5) of each vaccinated group (A, B, C and D) with higher lethal dose (10³ DLD₅₀) of virulent DEV in 1 mL volume, subcutaneously. Five birds in the control group were kept without any challenge infection. All the birds were kept under observation until 21 days post challenge (dpc) for any clinical signs or death due to challenge viral infection. The number of deaths for each group was recorded and the percent protection was determined using the formula: (number of birds survived/total number of birds) x 100.

2.13. Viral shedding in challenged birds

Cloacal swabs were collected in sterile tubes containing 2 mL PBS with antibiotics from vaccinated and control birds on the 2^nd, 4^th, 6^th and 8^th dpc. The samples were clarified by centrifugation at 5,000 rpm for 15 min. The supernatant was used for DNA extraction using a DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s protocol. The presence of virus was determined by amplification of DNA polymerase gene of DEV in PCR (OIE Terrestrial Manual 2018).

2.14. Genomic sequencing and phylogenetic analysis

The complete genome sequence of the vaccine strain (DPvac/IVRI-19) was generated using next-generation sequencing technology. Eleven complete genome sequences were retrieved from the NCBI GenBank public database for a comparative study. Due to the unusual gaps and high number of variants in some regions of the genome, the NC_013036 genome sequence (Li et al. 2009) was used as a reference and the genomic coordinate in this study. As already described in our recent publication (Dandapat et al. 2022), the complete genome sequence of virulent (DEV/India/IVRI-2016) and attenuated (DPvac/IVRI-19) DEV strain was generated and aligned to NC_013036 reference genome. Further, the differences in comparison with NC_013036 were extracted to create variants. Gene annotations of virulent and attenuated strains were performed using GATU software. DEV/India/IVRI-2016 and DPvac/IVRI-19 genome sequences were aligned with other complete genome sequences available in NCBI GenBank using MEGA 10 software (Tamura et al. 2021) and a consensus phylogenetic tree was generated using NG Phylogeny software. Further, a phylogenetic tree was also generated based on comparison of the sequences of DNA polymerase gene of Indian isolates with the other DEV strains available in NCBI GenBank.

2.15. Statistical analysis

All data were presented as mean with standard error. The data analysis and figures were made using GraphPad prism software (version 8.0.2). LTT and VNT assay data were analysed using one-way ANOVA. Flow cytometry data were analysed with two tailed t-test. Data were considered statistically significant if P value is *p < 0.05 or **p < 0.01 or ***p < 0.001 or ****p < 0.0001.

3. Results

3.1. Adaptation and serial propagation of DEV in CEF cell culture

The virus infected CEF cells were observed routinely under an inverted microscope for appearance of obvious CPE. The characteristic CPE like rounding of cells, syncitia formation, aggregation of cells forming an appearance of bunch of grapes, detachment of cells from the surface forming more gaps in the monolayer etc. were observed (Figure 1). During initial passages the optimum CPE was observed after 96 hpi and this was reduced to 48-72 hpi with increase in the passage levels. Presence of DEV DNA in the infected cell culture fluid at different passage levels was demonstrated by PCR amplification of the DNA polymerase gene. The PCR products (amplicons) of 446 bp in size were visualized in agarose gel electrophoresis as expected (Figure 2). The localization of virus by IIFT revealed the presence of DEV antigens in the virus infected CEF cell culture showing apple green fluorescence, whereas there was no fluorescence detected in the uninfected cell culture control (Figure 3).

Figure 1. Cytopathic effects in the virus infected CEF cell culture. (A) Control CEF cell monolayer; (B) CEF cell culture infected with DEV at 41^st passage, showing characteristic cytopathic effects like rounding of cells, syncytia formation, aggregation of cells resembling bunch of grapes, detachment of cells from the surface forming more gaps in the monolayer.

Figure 2. Detection of DEV in CEF cell culture. Amplification of DNA polymerase gene of DEV in the infected CEF cell culture at different passage levels showed an amplicon size of 446 bp.

Figure 3. Demonstration of viral antigens in the infected CEF culture by indirect immunofluorescence test. (A). Control CEF culture showing no fluorescence; (B) localization of DEV viral antigens in the infected CEF culture was revealed by appearance of apple green fluorescence.

3.2. Virus growth kinetics and titers at different passage levels

Our results on growth kinetics of the virus at P35 revealed that there was an increase in virus titer from 12 to 72 hpi, with a peak titre of 10^7.2 TCID₅₀/mL, followed by a decline in virus titer (10^6.49 TCID₅₀/mL) at 96 hpi (Figure 4). Further, it was found that the virus titers increased gradually with the increase in passage levels and ultimately reached 10^6.5 TCID₅₀/0.1 mL or 10^7.5 TCID₅₀/mL at the 41^st passage (Figure 5)

Figure 4. Growth kinetics of DEV in the CEF cell culture. The virus was quantified at various time points by determining the TCID₅₀ that showed an increase in virus titre up to 72 hpi, followed by a decrease in titre at 96 hpi.

Figure 5. Virus titres at different passage levels. The virus titre (log₁₀ TCID₅₀/0.1 mL) in the infected CEF cell culture showed a gradual increase pattern with the increase of passage levels.

3.3. Safety and stability of the cell culture attenuated DEV vaccine

In the safety test, the vaccinated ducklings did not show any clinical signs and no death was observed until 21 dpi. Thus, the DPvac/IVRI-19 was found to be avirulent and completely safe to the ducklings indicating that the virus has been attenuated. Further, the serial back passaging of the vaccine for 6 times in ducklings did not show any kind of clinical signs or lesions even up to 21 dpi after 6^th passage. The results demonstrated the stability of vaccine virus maintaining the attenuation property without reversion to virulence even after 6 passages in the natural host.

3.4. Virus neutralizing antibody levels in the vaccinated ducklings

The serum samples collected from immunized birds at 7, 14, 21 dpi demonstrated gradual increase in neutralizing antibody titre with significant inhibition of virus replication against 200 TCID₅₀ of virus. The virus neutralizing titers at 21 dpi in the vaccinated groups (B, C and D) reached up to 2^4.3, 2^4.6, 2^4.6, respectively, whereas the serum samples of group A, which had received vaccine dose of 10^2.5 TCID₅₀ showed the titer of 2^3.3 (Figure 6). Virus control showed distinct CPE with 200 TCID₅₀ and 20 TCID₅₀ of virus, while the cell control had a distinctive monolayer with no CPE.

Figure 6. Virus-neutralizing antibody titres. The mean protective antibody titre reached up to 2^3.3 in group A and 2^4.3, 2^4.6, 2^4.6, in group B, C and D, respectively. There was significant difference ****p < 0.0001 between immunized and control groups. Data were analyzed by one-way ANOVA.

3.5. Cellular immune response in the vaccinated ducklings

In the vaccinated groups, the lymphocyte blastogenic response to the DEV antigen in terms of SI gradually increased from 14 dpi (Figure 7). All the immunized groups showed significantly higher antigen-specific lymphoproliferative response compared to control. Flow cytometry analysis of PBMCs at 7, 14 and 21 dpi revealed the significant increase in the levels of CD4+ and CD8+ T lymphocytes in the vaccinated groups compared to control group. There was a gradual enhancement of the T lymphocyte populations with an increase in time. In the vaccinated groups, the highest level of CD4+ and CD8+ T cells was noticed at 21 dpi, and it was statistically significant (p < 0.0001) as compared to control (Figure 8). However, no significant difference was noticed in the control group at all time points.

Figure 7. Lymphocyte proliferation assay. Antigen-specific lymphocyte blastogenic response in the PBMC of different groups of birds (n = 10) at 7, 14, and 21 dpi was determined by MTT colorimetric assay. The data were presented as mean stimulation indices for each group. The statistical significance of the vaccinated group compared to control group is denoted as ****(p < 0.0001).

Figure 8. Flow cytometry analysis of CD4+ and CD8+ T-cells. T-lymphocyte subsets population in PBMC of the ducklings of different groups at 7, 14, and 21 dpi were determined by flow cytometry analysis. The mean percent of CD4+ and CD8+ T lymphocytes showed a significant difference of ****p < 0.0001 between the immunized and control group. Statistical analysis was performed using a two-tailed t-test.

3.6. Protective efficacy of the cell culture attenuated DEV vaccine

In potency testing all the birds of vaccinated groups (B, C, and D, which had received vaccine dose of 10^3.5 TCID₅₀ or more) survived challenge infection of higher lethal doses, i.e. 10² and 10³ DLD₅₀ and did not show any clinical sign or death until 21dpc, thus afforded 100% protection, whereas in the group A, which received the vaccine dose of 10^2.5 TCID₅₀, 2 out of 10 birds died with challenge dose of 10² DLD₅₀ (80% protection) and 2 out of 5 birds died with challenge dose of 10³ DLD₅₀ (60% protection). On the contrary, all the 10 ducklings of the unvaccinated group-E, which received challenge dose of 10² DLD₅₀, died within 5-6 dpc showing typical clinical signs of duck plague.

3.7. Viral shedding

PCR amplification of virus using the cloacal swab samples revealed positive viral shedding in the vaccinated groups (A, B, C and D) and control birds on 2 dpc. At 4 dpc, a significant reduction in viral shedding was noticed in the vaccinated group as compared to the control. Viral shedding was completely nil after 6 dpc in group B, C and D, whereas 13% positive viral shedding noticed in group A, which had received the vaccine dose of 10^2.5TCID₅₀ (Table 1). However, unvaccinated and challenged control birds showed constant viral shedding and there was no viral shedding observed in unvaccinated and unchallenged birds (group E).

Table 1. Viral shedding pattern in different groups of vaccinated ducks after challenge infection with virulent DEV.

Groups	Vaccine dose	Days post challenge (dpc)
		2 dpc	4 dpc	6 dpc	8 dpc
Group A	10^2.5 TCID50	15/15	10/15	4/15	2/15
Group B	10^3.5 TCID50	14/15	6/15	0/15	0/15
Group C	10^4.5 TCID50	13/15	5/15	0/15	0/15
Group D	10^5.5 TCID50	13/15	5/15	0/15	0/15
Group E	Unvaccinated	10/10	10/10	died	died
Group E (without challenge)	Unvaccinated	0/5	0/5	0/5	0/5

The presence of virus in the cloacal swabs collected at 2^nd, 4^th, 6^th and 8^th dpc was determined by amplification of DNA polymerase gene of DEV in PCR. The virus clearance was observed by 6 dpc in the vaccinated groups (B, C & D), whereas the unvaccinated challenged birds showed virus shedding until death.

3.8. Genomic characterization of DPvac/IVRI-19

One hundred seventy-eight nucleotide variations were found in the vaccine candidate in comparison with the reference genome NC_013036 among which 159 are genetic variants with synonymous and non-synonymous mutations as compared to the reference strain. A comparison of vaccine strain DPvac/IVRI-19 (MZ911871) with the virulent field isolate DEV/India/IVRI-2016 (MZ824102) revealed 9 non-synonymous mutations, 10 silent/synonymous mutations and a replacement of a single nucleotide in the UL43 gene resulted in a stop signal and premature termination of the coding gene (Table 2). The phylogenetic tree revealed that DEV/India/IVRI-2016 has certain unique mutations in them, forming a different cluster (Figure 9). Further, the phylogenetic tree of DNA polymerase gene revealed that DEV/India/IVRI-2016 is more closely related to the prevailing Indian DEV isolates (Figure 10).

Figure 9. Phylogenetic analysis of the DEV genome. The phylogenetic analysis of DEV/India/IVRI-2016 was performed against other DEV isolates (n = 11) available in the NCBI database by NGPhylogeny.fr., which suggested that the Indian strains (DEV/India/IVRI-2016 and DPvac/IVRI-19) showed an evolutionary relationship with other DEV isolates and they have certain unique mutations forming a separate cluster.

Figure 10. Phylogenetic analysis of the DNA polymerase gene of DEV strains. It revealed that DEV/India/IVRI-2016 is more closely related to the prevailing Indian DEV isolates.

Table 2. Mutations at different positions in the coding genes of DPvac/IVRI-19 vaccine strain as compared with the DEV/India/IVRI-2016 virulent isolate.

Mutation	Position	Gene	Attenuated	Virulent
Non synonymous mutation	14708	UL49 (nt)	A	G
		(aa)	H	R
	40187	UL37/36 (nt)	T	C
		(aa)	V	A
	74310	UL24 (nt)	T	G
		(aa)	H	Q
	75384	UL23 (nt)	G	T
		(aa)	R	S
	100534	UL9 (nt)	T	C
		(aa)	Y	H
	104511	UL6 (nt)	A	G
		(aa)	S	W
	142736	US7 (nt)	A	T
		(aa)	Q	L
	142738	US7 (nt)	G	T
		(aa)	G	C
Nonsense mutation	26920	UL43 (nt)	T	A
		(aa)	(.)	A
Synonymous mutation	2832	LORF11 (nt)	C	C
	27733	UL42 (nt)	C	T
	41834	UL36 (nt)	G/C	A
	13199	UL50 (nt)	A	C
	73374	UL24 (nt)	A/G	G
	84915	UL19 (nt)	C	T
	100887	UL9 (nt)	C	A
	101757	UL8 (nt)	A/G	A
	119424	UL3 R (nt)	A	G
	134555	SORF3/US2 (nt)	T	C

Comparison between whole genome sequences of DPvac/IVRI-19 and DEV/India/IVRI-2016 showing mutations and amino acid changes in different genes at different positions.

4. Discussion

Duck plague causes severe morbidity and mortality, posing a severe threat to the duck industry with huge economic losses worldwide (Dhama et al. 2017). In India, disease prevalence has also been increasing with the timeline (Rajan et al. 1980; Dhama et al. 2017; Neher et al. 2019; Pazhanivel et al. 2019). Vaccination is the most efficient and economical way to control and prevent the devastating effects of the duck enteritis virus (DEV). The currently available commercial vaccine in India is a chicken embryo-adapted live attenuated DP vaccine, prepared from a foreign strain (Holland strain/Jansen strain), which is being used for more than last four decades. In general, the chicken embryo-adapted vaccines are cumbersome to produce in a large scale. Few studies have reported the outbreaks of DP even in the vaccinated flocks, indicating the need for an improved vaccine with better efficacy (Rajan et al. 1980; Rani and Muruganandan 2015). The vaccination failure may be attributed to continuous genetic and antigenic changes of the circulating DPV that impairs the efficacy of vaccine (Kulkarni et al. 1998). Keeping in view all such limitations with the current vaccine, the present study aimed at the strategic approach to develop a CEF cell culture based live attenuated DP vaccine using an Indian field isolate (DEV/India/IVRI-2016). In the process of achieving the target, the virus was isolated and fully characterized along with whole genome analysis (MZ824102) as mentioned in our previous studies (Panickan et al. 2021; Dandapat et al. 2022). The virus was initially propagated in duck embryos (P15), followed by DEF (P15) and finally, the DEF passaged virus was adapted in CEF cell culture and serially propagated up to 41 passages (P41) to achieve the attenuation of the virus. Propagation of DEV in primary CEF was confirmed by appearance of characteristic CPE comprising of rounding and clumping of cells, marked cytoplasmic granulations, formation of large number of intracellular vacuoles and finally cell death. Confirmation of virus in CPE-positive cultures was accomplished using PCR by amplifying DNA polymerase gene of DEV. For detection of DEV nucleic acid, DNA polymerase gene was targeted by various workers because of highly conserved nature (Xuefeng et al. 2008). We further confirmed localization of DEV antigens in CEF cell culture by IIFAT using DEV-specific antibodies, displaying the emission of apple green fluorescence in the infected cell culture. With respect to viral growth characteristics in CEF, during initial few passages the CPE started to appear after 96 hpi, but as the passage level increased, CPE was observed within 48-72 hpi. More than 80% CPE was observed at 60 to 72 hpi and after 72 hpi cell monolayer detached from the surface of the culture vessels, which was also corroborated by the maximum virus titre at this time. The viral growth kinetics at P35 showed an increase in virus titer from 12 hpi to 72 hpi, with a peak titre of 10^7.2 TCID₅₀/mL at 72 hpi, followed by a decline in virus titer (10^6.49 TCID₅₀/mL) at 96 hpi. The similar observation of appearance of CPE has been reported while a DP vaccine virus was adapted to grow in CEF cell culture (Doley et al. 2013). In the present study, the virus was well adapted to the CEF cells, which was evident from the gradual increase of virus titers with the increase in passage levels that ultimately reached 10^6.5 TCID₅₀/0.1 mL or 10^7.5 TCID₅₀/mL at the 41^st passage.

Safety and potency tests of the vaccine candidate by animal inoculation are the important parameters recommended by OIE as well as Indian Pharmacopeia to confirm attenuation and to establish the efficacy of the vaccine. We have checked the safety of the CEF cell culture passaged virus at P41 (DPvac/IVRI-19) by subcutaneously administering ducklings with 10 times of the effective vaccine dose (10×). No clinical sign was found in immunized ducklings until 21 dpi, indicating that the vaccine virus was avirulent, safe and completely attenuated. However, an important safety concern with live attenuated vaccines is the chance of reversion to a virulent form under natural conditions. Our experiment of serial back passaging of the vaccine in ducklings did not reveal any clinical sign or adverse effect, which demonstrated the stability of the attenuated virus without reversion to virulence even after 6 passages in the natural host. After confirming safety, an immunization trial was conducted in ducklings with different vaccine doses (10^2.5, 10^3.5, 10^4.5 and 10^5.5 TCID₅₀)/mL of DPvac/IVRI-19, in four groups (A, B, C, D, respectively), and the fifth group E served as unvaccinated control. The serum samples of the vaccinated groups (B, C and D) showed comparatively higher virus-neutralizing titers of 2^4.3, 2^4.6 and 2^4.6 respectively, against 200 TCID₅₀ of the virus than group A (2^3.3). The amount of neutralizing antibody correlated to percent protection. The antibody levels with VNT titer of 1:16 to 1:32 was found in groups B, C and D, in which protection was 100%. However, immunity against DEV infection is primarily based on cellular immunity rather than humoral and low levels of neutralizing antibodies are produced (Dardiri 1975; Kulkarni et al. 1998).

In this study, the level of immunity developed in the vaccinated ducks was sufficient to provide complete protection. Apart from protective antibody levels, we also evaluated the CMI response by lymphocyte proliferation and enumeration of CD4+ and CD8+ T cells. It is well known that both CD4+ and CD8+ play a vital role in herpesvirus clearance (Freeman et al. 2012). In addition, CD4+ T cells aids in the functional assistance of CD8+ T cells and help in the antiviral immunity (Huang et al. 2014). As the live attenuated vaccine enables the efficient MHC class I presentation of antigens that can stimulate CD8+ T lymphocyte responses (Sevimli et al. 2012). Our data suggest that there was a gradual increase of both CD4+ and CD8+ T cells after immunization with different doses of DPvac/IVRI-19, which is in agreement with the findings of Huang et al. (2014). The significant differences (p < 0.001) between the immunized and control groups correlate with the protection against challenge infection.

Protection against virulent pathogens is the best criterion to assess the efficacy of vaccines. After challenging the ducks with 100 or 1000 DLD₅₀ of local virulent DEV strain by subcutaneous injection, the ducks were observed for clinical signs for 21 days. All the ducks of groups (B, C, & D) that received the vaccine dose of 10^3.5 TCID₅₀ or more were completely protected and showed no clinical signs, with all ducks surviving until 21 days, indicating 100% protection against both the lethal doses of challenge virus. However, group A vaccinated birds showed 80% and 60% protection against challenge infection with 10² and 10³ DLD_50, respectively. In contrast, unvaccinated control birds (group E) died with typical clinical signs of DP upon challenge virus infection. Thus, a single subcutaneous immunization with the vaccine dose of 10^3.5 TCID₅₀ was optimum, which afforded 100% protection against challenge virus infection. The route of vaccination also influences vaccine immunogenicity, the kinetics of the attenuated Chinese vaccine strain revealed that subcutaneous administration results in greater vaccine distribution in tissues as compared to oral or nasal administration (Qi et al. 2009; Huang et al. 2014). Overall, the study demonstrated that cell-mediated immunity along with neutralizing antibody response played an important role in blocking the replication of challenge virus, reducing viral shedding, and eventually afforded solid protection in the vaccinated birds. In addition, the vaccinated unchallenged flock was maintained for more than a year to evaluate the persistence of protective immunity. During the observation period, the birds did not exhibit any adverse effect or clinical signs.

Further, the assembled DPvac/IVRI-19 genome sequence (MZ911871) was 158kbp, the same length as that of parental virus DEV/India/IVRI-2016 (MZ824102). Comparative genome analysis revealed that the attenuated virus genome sequence had no deletions or insertions, but nucleotide substitutions led to an amino acid change in the open reading frames in UL49, UL37/36, UL24, UL23, UL9, UL6, and US7 along with 10 synonymous mutations. In addition, the nonsense mutation by substituting one base (A→T) in the UL43 gene resulted in a stop codon, with premature termination of gene. Alteration in the UL43 gene does not affect the replication and growth of the virus (Powers et al. 1994). The supportive findings with Equine Herpesvirus I revealed the novelty of the UL43 gene in negative regulation of MHC I antigen presentation (Huang et al. 2015). Our data suggest that the mutations in the several ORFs in DPvac/IVRI-19 genome contribute to the loss of virulence and attenuation of the virus. Further, advance studies are required for establishing the predominant role of UL43 gene in duck plague which may help in the target inhibition of virus replication and enhancement of host immune response. In addition, phylogenetic analysis showed an evolutionary relationship among basal branches with other DEV isolates. The attenuated virus sustained a significant number of mutations evolving from the parental virus and distantly related to the other strains of DEV. Further, phylogenetic analysis of the DNA polymerase gene revealed its close relationship with the prevalent strain of DEV in India.

5. Conclusions

The DPvac/IVRI-19 attenuated vaccine was found to be safe and efficacious inducing both arms (cell mediated and humoral immune response) of the immune system, even though virus-neutralizing antibody titre seems to be moderate. The significant predominance of cellular immune response afforded solid protection in the immunized ducks against high lethal dose of challenge infection. Since, vaccination is the only means of prevention of DP, usage of DPvac/IVRI-19 cell culture vaccine will have advantages over the existing chicken embryo adapted duck plague vaccine presently being used in India and may be also in many other countries. The new vaccine candidate has been prepared from a local DEV field strain; hence it may be more effective against the presently circulating field strain while eliciting long lasting robust protective immunity. However, further studies are required to evaluate the field safety and effectiveness of DPvac/IVRI-19, compared to existing vaccine.

Author contributions statement

Satyabrata Dandapat: Conception and design, supervision, funding acquisition, investigation, interpretation of data, drafting and revising the manuscript; Suresh Bindu: Experiment design, investigation, analysis and interpretation of data, drafting and revision; Gaurav Kumar Sharma: Investigation, data analysis, manuscript editing; Sivasankar Panickan: Experiment design, data analysis, drafting and revision; Sukdeb Nandi: Investigation, manuscript editing; G. Saikumar; Conceptualization, manuscript editing; Kuldeep Dhama: Investigation, manuscript drafting and revision. All authors have final approval of the version of the manuscript to be published and that all authors agree to be accountable for all aspects of the work.

Acknowledgements

The authors are highly thankful to the Director, ICAR-Indian Veterinary Research Institute and Indian Council of Agricultural Research for providing the funds and the facilities to carry out this work.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The data that support the findings of this study are available in supplementary information or from the corresponding author upon reasonable request.

Additional information

Funding

This study was supported through an institute research project of ICAR-Indian Veterinary Research Institute, Izatnagar, funded by the Indian Council of Agricultural Research (ICAR), New Delhi, India.