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

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 107.5 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.

Differential Virus-Specific IFN-Gamma Producing T Cell Responses to Marek's Disease Virus in Chickens With B19 and B21 MHC Haplotypes.

Marek's disease virus (MDV), the etiologic agent for Marek's disease (MD), causes a deadly lymphoproliferative disease in chickens. Causes of the well-documented association between genetically defined lines of chicken and resistance to MD remain unknown. Here, the frequencies of IFN-gamma producing pp38 and MEQ-specific T cell responses were determined in line N (B21 haplotype; MD-resistant) and line P2a (B19 haplotype, MD-susceptible) chickens after infection with vaccine and/or virulent (RB1B) strains of MDV using both standard ex vivo and cultured chIFN-gamma ELISPOT assays. Notably, MDV infection of naïve and vaccinated MD-resistant chickens induced higher frequencies of IFN-gamma producing MDV-specific T cell responses using the cultured and ex vivo ELISPOT assay, respectively. Remarkably, vaccination did not induce or boost MEQ-specific effector T cells in the susceptible chickens, while it boosted both pp38-and MEQ-specific response in resistant line. Taken together, our results revealed that there is a direct association between the magnitude of T cell responses to pp38 and MEQ of MDV antigens and resistance to the disease.

Development of a simple and rapid immunochromatographic strip test for detecting duck plague virus antibodies based on gI protein.

A colloidal gold strip (CGS) for detecting antibodies to duck plague virus (DPV) was developed. Colloidal gold-labeled DPV gI protein and goat anti-rabbit IgG were dispensed on a conjugate pad as tracers. The recombinant DPV gI protein and rabbit IgG were used as capture reagents at the test line and control line, respectively. The detection limit of this assay was 1:256. Additionally, the CGS did not react with antisera from other common duck diseases, only reacting with anti-DPV serum and yielding a specific and strong red signal. 123 serum samples were tested by CGS and enzyme-linked immunosorbent assay (ELISA), and the results showed good agreement. The CGS test results can be observed in 15 min with the naked eye, should be suitable for clinical testing and large-scale detection.

Evaluation of Factors That Influence Dose Variability of Marek's Disease Vaccines.

Marek's disease (MD) vaccines are cell-associated and require special handling and care during administration. Vaccine dose is evaluated by plaque assay and is indicated as the number of plaque-forming units (PFUs) per dose. The objectives of this study were to evaluate the dose variability within each vial of MD vaccines and to assess those factors (from both manufacturing and handling and administration of the vaccine) that could affect vaccine dose variability. Three experiments were conducted. Experiment 1 was to evaluate dose variability in 36 MD vaccine vials and the effect of manufacturing factors on dose variability. Vaccines were titrated 10 times. Dose variability was measured as the coefficient of variability (CV) calculated as standard deviation divided by average PFU and multiplied by 100. Our results showed that all evaluated vaccines had levels of CV ranging from 10% to 34%. Variability existed regardless of manufacturer, vaccine serotype, and batch. Experiment 2 was conducted to evaluate the effect of infectivity rate (IR) on CV. IR was artificially reduced by adding noninfected chicken embryo fibroblast to the reconstituted vaccine before titration. Our results showed that decreased IR results in higher CV. Experiment 3 was to evaluate the handling and administration factors (time and mixing during administration) on CV. Our results showed that CV tends to increase with time and that this effect is more remarkable if vaccines were not mixed. Our study emphasizes the relevance of proper handling of MD vaccines and shows that dose variability can jeopardize the uniformity of vaccination in a flock and therefore the success of vaccination.

Evaluación de factores que influyen en la variabilidad de las dosis de las vacunas contra la enfermedad de Marek. Las vacunas contra la enfermedad de Marek (MD) están asociadas a células y requieren un manejo y cuidado especiales durante la administración. La dosis de la vacuna se evalúa mediante un ensayo de placa y se indica como el número de unidades formadoras de placa (UFP) por dosis. Los objetivos de este estudio fueron evaluar la variabilidad de la dosis dentro de cada vial de vacunas contra la enfermedad de Marek y evaluar los factores (tanto de fabricación como de manipulación/administración de la vacuna) que podrían afectar la variabilidad de la dosis de la vacuna. Se realizaron tres experimentos. El experimento número 1 consistió en evaluar la variabilidad de la dosis en 36 viales de vacuna de Marek y el efecto de los factores de fabricación en la variabilidad de la dosis. Las vacunas fueron tituladas 10 veces. La variabilidad de la dosis se midió como el coeficiente de variación (CV) calculado como desviación estándar dividido por las UFP promedio y multiplicado por 100. Nuestros resultados mostraron que todas las vacunas evaluadas tenían coeficientes de variación que variaban del 10% al 34%. La variabilidad existía independientemente del fabricante, el serotipo de la vacuna y el lote. El experimento número 2 se realizó para evaluar el efecto de la tasa de infectividad (IR) en el coeficiente de variación. La tasa de infectividad se redujo artificialmente mediante la adición de fibroblastos de embrión de pollo no infectados a la vacuna reconstituida antes de la valoración. Los resultados mostraron que la disminución en la tasa de infectividad resulta en mayores coeficientes de variación. El experimento número 3 consistió en evaluar los factores de manipulación y administración (tiempo y mezclado durante la administración) sobre los coeficientes de variación. Nuestros resultados mostraron que el coeficiente de variación tiende a aumentar con el tiempo y que este efecto es más notable si las vacunas no se mezclan. Este estudio enfatiza la relevancia del manejo adecuado de las vacunas contra la enfermedad de Marek y muestra que la variabilidad de la dosis puede poner en peligro la uniformidad de la vacunación en una parvada y por lo tanto el éxito de la vacunación.

Galectin-1 induces immune response and antiviral ability in Cherry Valley ducks after duck plague virus infection.

Galectin-1, as a typical animal galactose-binding protein, it is found on the cell surface and in the extracellular matrix. Cloning the full-length coding sequence of galectin-1 from the spleens of Cherry Valley ducks revealed that the coding sequence of duck galectin-1 (duGal-1) comprises 405 bp, encoding 134 amino acids. Homologic analysis revealed its amino acid sequence is most identical to that of Anas platyrhynchos (98.8%) followed by Gallus gallus. Quantitative real-time PCR analysis indicated that duGal-1 mRNA is broadly expressed in healthy Cherry Valley duck tissues, primarily in the heart and trachea but minimally in the lung and skin. Meanwhile, the duGal-1 expression is slightly upregulated in the infected liver and spleen. Furthermore, the expression levels of ISGs (Mx, PKR, OAS) and some cytokines such as IFN-α, IL-1ß, IL-2, are up-regulated to varying degrees after overexpression the duGal-1, In contrast, Knockdown of duGal-1 found that the expression levels of ISGs and some inflammatory cytokines were down-regulated. Antiviral assay showed that duGal-1 could inhibit viral replications early during infection. This is the first study of the cloning, tissue distribution, and antiviral immune responses of duGal-1, and findings imply it is involved in the early stages of antiviral innate immune responses to duck plague virus infections in ducks.

Isolation and Selection of Duck Primary Cells as Pathogenic and Innate Immunologic Cell Models for Duck Plague Virus.

Duck plague virus (DPV) is a representative pathogen transmitted among aquatic animals that causes gross lesions and immune inhibition in geese and ducks. The mechanism of organ tropism and innate immune evasion of DPV has not been completely deciphered due to a lack of cell models to study the innate immune manipulation and pathogenicity of aquatic viruses. In the present study, we isolated five types of duck primary cells [duck embryo fibroblasts (DEFs), neurons, astrocytes, peripheral blood mononuclear cells (PBMCs), and monocytes/macrophages] to identify appropriate cell models for DPV, using tropism infection and innate immunologic assays. Cells responded differently to stimulation with DNA viruses or RNA virus analogs. DPV infection exhibited broad tropism, as the recombinant virulent strain (CHv-GFP) infected DEFs, neurons, astrocytes, and monocytes/macrophages, but not the PBMCs, as the expression of EGFP was negligible. The basal levels of innate immunity molecules were highest in monocytes/macrophages and lower in DEFs and astrocytes. Conversely, the titer and genomic copy number of the attenuated virus strain was higher in DEFs and astrocytes than in neurons and monocytes/macrophages. The titer and genomic copy number of the attenuated virus strain were higher compared with the virulent strain in DEFs, neurons, and astrocytes. The innate immune response was not significantly induced by either DPV strain in DEFs, neurons, or astrocytes. The virulent strain persistently infected monocytes/macrophages, but the attenuated strain did so abortively, and this was accompanied by the phenomenon of innate immune inhibition and activation by the virulent and attenuated strains, respectively. Blockage of IFNAR signaling promoted replication of the attenuated strain. Pre-activation of IFNAR signaling inhibited infection by the virulent strain. The selection assay results indicated that induction of innate immunity plays an essential role in controlling DPV infection, and monocytes/macrophages are an important cell model for further investigations. Our study provided practical methods for isolating and culturing duck primary cells, and our results will facilitate further investigations of organ tropism, innate immune responses, latent infection, and the effectiveness of antiviral drugs for treating DPV and potentially other aerial bird pathogens.

Why the evolution of vaccine resistance is less of a concern than the evolution of drug resistance.

Vaccines and antimicrobial drugs both impose strong selection for resistance. Yet only drug resistance is a major challenge for 21st century medicine. Why is drug resistance ubiquitous and not vaccine resistance? Part of the answer is that vaccine resistance is far less likely to evolve than drug resistance. But what happens when vaccine resistance does evolve? We review six putative cases. We find that in contrast to drug resistance, vaccine resistance is harder to detect and harder to confirm and that the mechanistic basis is less well understood. Nevertheless, in the cases we examined, the pronounced health benefits associated with vaccination have largely been sustained. Thus, we contend that vaccine resistance is less of a concern than drug resistance because it is less likely to evolve and when it does, it is less harmful to human and animal health and well-being. Studies of pathogen strains that evolve the capacity to replicate and transmit from vaccinated hosts will enhance our ability to develop next-generation vaccines that minimize the risk of harmful pathogen evolution.

US10 Protein Is Crucial but not Indispensable for Duck Enteritis Virus Infection in Vitro.

To investigate the function of the duck enteritis virus (DEV) tegument protein US10, we generated US10 deletion and revertant mutants (ΔUS10 and US10FRT) via two-step RED recombination based on an infectious BAC clone of DEV CHv-BAC-G (BAC-G). In multistep growth kinetic analyses, ΔUS10 showed an approximately 100-fold reduction in viral titer, while the genome copies decreased only 4-fold compared to those of BAC-G. In one-step growth kinetic analyses, there were no significant differences in genome copies among BAC-G, ΔUS10 and US10FRT, but ΔUS10 still showed a 5- to 20-fold reduction in viral titer, and the replication defect of ΔUS10 was partially reversed by infection of US10-expressing cells. The transcription levels of Mx, OASL, IL-4, IL-6 and IL-10 in ΔUS10-infected duck embryo fibroblasts (DEFs) were significantly upregulated, while TLR3 was downregulated compared with those in BAC-G-infected DEFs. Taken together, these data indicated that US10 is vital for DEV replication and is associated with transcription of some immunity genes.

Molecular cloning and functional characterization of duck DDX41.

DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 (DDX41), a receptor belonging to DExD/H-box helicase family, acts as an intracellular DNA sensor and induces type I IFN production in mammals and fish. However, the function of avian DDX41 in innate immune response is still unknown. In this study, the full-length duck DDX41 (duDDX41) cDNA sequence was cloned for the first time and encoded a putative protein of 618 amino acid residues which showed the high sequence similarity with both zebra finch and chicken DDX41s. The duDDX41 mRNA was widely distributed in all tested tissues, especially the cerebrum, cerebellum, and liver. Overexpression of duDDX41 triggered the activation of transcription factors IRF1 and NF-κB, as well as IFN-ß expression in DEFs. The DEADc domain of duDDX41 played an extremely vital role in duck type I IFN signaling pathway. Knockdown of duDDX41 by siRNA silencing dramatically decreased IFN-ß expression stimulated by poly(dA:dT) or duck enteritis virus (DEV). In addition, the replication of DEV was significantly inhibited in duDDX41-expressed DEFs and was enhanced in DDX41 knockdown DEFs. These results suggest that DDX41 is an important cytosolic DNA sensor and plays a crucial role in duck antiviral innate immune response.

The Application of NHEJ-CRISPR/Cas9 and Cre-Lox System in the Generation of Bivalent Duck Enteritis Virus Vaccine against Avian Influenza Virus.

Duck-targeted vaccines to protect against avian influenza are critically needed to aid in influenza disease control efforts in regions where ducks are endemic for highly pathogenic avian influenza (HPAI). Duck enteritis virus (DEV) is a promising candidate viral vector for development of vaccines targeting ducks, owing to its large genome and narrow host range. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a versatile gene-editing tool that has proven beneficial for gene modification and construction of recombinant DNA viral vectored vaccines. Currently, there are two commonly used methods for gene insertion: non-homologous end-joining (NHEJ) and homology-directed repair (HDR). Owing to its advantages in efficiency and independence from molecular requirements of the homologous arms, we utilized NHEJ-dependent CRISPR/Cas9 to insert the influenza hemagglutinin (HA) antigen expression cassette into the DEV genome. The insert was initially tagged with reporter green fluorescence protein (GFP), and a Cre-Lox system was later used to remove the GFP gene insert. Furthermore, a universal donor plasmid system was established by introducing double bait sequences that were independent of the viral genome. In summary, we provide proof of principle for generating recombinant DEV viral vectored vaccines against the influenza virus using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Duck stimulator of interferon genes plays an important role in host anti-duck plague virus infection through an IFN-dependent signalling pathway.

The human stimulator of interferon gene (STING) is an important molecule in innate immunity that stimulates type I interferon (IFN) production. However, the role of duck STING (duSTING) in innate immunity has yet to be explained. In this study, the full length of the duSTING cDNA sequence (1149bp), which encodes 382 amino acid (aa) residues, was reported and showed the highest sequence similarity with chicken STINGs. The phylogenetic analysis based on STING aa showed that duSTING was grouped onto the birds clade. According to the tissue distribution spectrum analysis, duSTING was highly present in the bursa of Fabricius, glandular stomach, liver, pancreas, and small intestine of ducklings, as well as in the blood and pancreas of the adult duck. DuSTING mainly colocalized with the endoplasmic reticulum (ER) and mitochondria in transfected Baby Hamster Syrian Kidney (BHK21) and duck embryo fibroblasts (DEF) cells by an indirect immunofluorescence assay. The transfection of the DEFs with duSTING activated NF-κB, which induced the transcription of IFN-ß, and the activated IFN induced the interferon-stimulated response element (ISRE). Furthermore, the overexpression of duSTING significantly upregulated the mRNA level of duck IFN-ß and IFN-stimulated genes (ISGs), such as duMx and duOASL and inhibited the replication of the double-stranded DNA duck plague virus (DPV) in vitro. In addition, the knockdown of endogenous duSTING by shRNA significantly reduced the poly (I:C) (pIC), poly (dA:dT), and Tembusu virus (TMUV), induced IFN-ß production and significantly promoted DPV replication in vitro. In general, these data demonstrate that duSTING is vital for duck type I interferon induction and plays an important role in the host defence of DPV infection.

Cross-species antiviral activity of goose interferon lambda against duck plague virus is related to its positive self-regulatory feedback loop.

Duck plague virus (DPV) is a virus of the Herpesviridae family that leads to acute disease with a high mortality rate in ducks. Control of the disease contributes to the development of poultry breeding. Type III IFN family (IFN-λs) is a novel member of the IFN family, and goose IFN-λ (goIFN-λ) is a newly identified gene whose antiviral function has only been investigated to a limited extent. Here, the cross-species antiviral activity of goIFN-λ against DPV in duck embryo fibroblasts (DEFs) was studied. We found that pre-treatment with goIFN-λ greatly increased the expression of IFN-λ in both heterologous DEFs and homologous goose embryo fibroblasts (GEFs), while differentially inducing IFNα- and IFN-stimulated genes. Additionally, a positive self-regulatory feedback loop of goIFN-λ was blocked by a mouse anti-goIFN-λ polyclonal antibody, which was confirmed in both homologous GEFs and goose peripheral blood mononuclear cells (PBMCs). The suppression of the BAC-DPV-EGFP by goIFN-λ in DEFs was confirmed by fluorescence microscopy, flow cytometry (FCM) analysis, viral copies and titre detection, which can be rescued by mouse anti-goIFN-λ polyclonal antibody incubation. Finally, reporter gene assays indicated that the cross-species antiviral activity of goIFN-λ against BAC-DPV-EGFP is related to its positive self-regulatory feedback loop and subsequent ISG induction. Our data shed light on the fundamental mechanisms of goIFN-λ antiviral function in vitro and extend the considerable range of therapeutic applications in multiple-poultry disease.

Construction of a highly efficient CRISPR/Cas9-mediated duck enteritis virus-based vaccine against H5N1 avian influenza virus and duck Tembusu virus infection.

Duck enteritis virus (DEV), duck tembusu virus (DTMUV), and highly pathogenic avian influenza virus (HPAIV) H5N1 are the most important viral pathogens in ducks, as they cause significant economic losses in the duck industry. Development of a novel vaccine simultaneously effective against these three viruses is the most economical method for reducing losses. In the present study, by utilizing a clustered regularly interspaced short palindromic repeats (CRISPR)/associated 9 (Cas9)-mediated gene editing strategy, we efficiently generated DEV recombinants (C-KCE-HA/PrM-E) that simultaneously encode the hemagglutinin (HA) gene of HPAIV H5N1 and pre-membrane proteins (PrM), as well as the envelope glycoprotein (E) gene of DTMUV, and its potential as a trivalent vaccine was also evaluated. Ducks immunized with C-KCE-HA/PrM-E enhanced both humoral and cell-mediated immune responses to H5N1 and DTMUV. Importantly, a single-dose of C-KCE-HA/PrM-E conferred solid protection against virulent H5N1, DTMUV, and DEV challenges. In conclusion, these results demonstrated for the first time that the CRISPR/Cas9 system can be applied for modification of the DEV genome rapidly and efficiently, and that recombinant C-KCE-HA/PrM-E can serve as a potential candidate trivalent vaccine to prevent H5N1, DTMUV, and DEV infections in ducks.

A Chinese Variant Marek's Disease Virus Strain with Divergence between Virulence and Vaccine Resistance.

Marek's disease (MD) virus (MDV) has been evolving continuously, leading to increasing vaccination failure. Here, the MDV field strain BS/15 was isolated from a severely diseased Chinese chicken flock previously vaccinated with CVI988. To explore the causes of vaccination failure, specific-pathogen free (SPF) chickens vaccinated with CVI988 or 814 and unvaccinated controls were challenged with either BS/15 or the reference strain Md5. Both strains induced MD lesions in unvaccinated chickens with similar mortality rates of 85.7% and 80.0% during the experimental period, respectively. However, unvaccinated chickens inoculated with BS/15 exhibited a higher tumor development rate (64.3% vs. 40.0%), but prolonged survival and diminished immune defects compared to Md5-challenged counterparts. These results suggest that BS/15 and Md5 show a similar virulence but manifest with different pathogenic characteristics. Moreover, the protective indices of CVI988 and 814 were 33.3 and 66.7 for BS/15, and 92.9 and 100 for Md5, respectively, indicating that neither vaccine could provide efficient protection against BS/15. Taken together, these data suggest that MD vaccination failure is probably due to the existence of variant MDV strains with known virulence and unexpected vaccine resistance. Our findings should be helpful for understanding the pathogenicity and evolution of MDV strains prevalent in China.

Evaluation and validation of reference gene stability during Marek's disease virus (MDV) infection.

Quantitative RT-PCR (qRT-PCR) is widely used in the study of relative gene expression in general, and has been used in the field of Marek's disease (MD) research to measure transcriptional responses to infection and/or vaccination. Studies in the past have either employed cellular ß-actin (BACT) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal reference genes, although the stability of their expression in the context of Marek's disease virus (MDV) infection has never been investigated. In the present study, we compared the stability of five reference genes (BACT, 28S RNA, 18S RNA, GAPDH, Peptidyl-prolyl-isomerase B [PPIB], a.k.a. cyclophilin B) as standard internal controls in chicken embryo fibroblast (CEFs) cultures infected with either MD vaccine or oncogenic MDV1 viruses. We further extend these analyses to reference gene stability in spleen lymphomas induced by infection of commercial broiler chickens with a very virulent plus MDV1 (vv+ TK-2a virus). Two excel based algorithms, (Bestkeeper and Normfinder) were employed to compare reference gene stability. Bestkeeper and Normfinder analysis of reference gene stability in virus- and mock-infected cells, showed that 28S RNA and PPIB displayed higher stability in CEF infections with either oncogenic or vaccine viruses. In addition, both Bestkeeper and Normfinder determined 28S RNA and PPIB to be the most stably-expressed reference genes in vivo in vv+ TK-2a-induced spleen lymphomas. Furthermore, Bestkeeper and Normfinder analyses both determined BACT to be the least stable reference gene during MDV infection of CEF with oncogenic viruses, vaccine viruses, as well as in vv+ TK-2a-induced spleen lymphomas.

Cross-Species Antiviral Activity of Goose Interferons against Duck Plague Virus Is Related to Its Positive Self-Feedback Regulation and Subsequent Interferon Stimulated Genes Induction.

Interferons are a group of antiviral cytokines acting as the first line of defense in the antiviral immunity. Here, we describe the antiviral activity of goose type I interferon (IFNα) and type II interferon (IFNγ) against duck plague virus (DPV). Recombinant goose IFNα and IFNγ proteins of approximately 20 kDa and 18 kDa, respectively, were expressed. Following DPV-enhanced green fluorescent protein (EGFP) infection of duck embryo fibroblast cells (DEFs) with IFNα and IFNγ pre-treatment, the number of viral gene copies decreased more than 100-fold, with viral titers dropping approximately 100-fold. Compared to the control, DPV-EGFP cell positivity was decreased by goose IFNα and IFNγ at 36 hpi (3.89%; 0.79%) and 48 hpi (17.05%; 5.58%). In accordance with interferon-stimulated genes being the "workhorse" of IFN activity, the expression of duck myxovirus resistance (Mx) and oligoadenylate synthetases-like (OASL) was significantly upregulated (p < 0.001) by IFN treatment for 24 h. Interestingly, duck cells and goose cells showed a similar trend of increased ISG expression after goose IFNα and IFNγ pretreatment. Another interesting observation is that the positive feedback regulation of type I IFN and type II IFN by goose IFNα and IFNγ was confirmed in waterfowl for the first time. These results suggest that the antiviral activities of goose IFNα and IFNγ can likely be attributed to the potency with which downstream genes are induced by interferon. These findings will contribute to our understanding of the functional significance of the interferon antiviral system in aquatic birds and to the development of interferon-based prophylactic and therapeutic approaches against viral disease.

Attenuated Salmonella Typhimurium delivery of a novel DNA vaccine induces immune responses and provides protection against duck enteritis virus.

DNA vaccines are widely used to prevent and treat infectious diseases, cancer and autoimmune diseases; however, their relatively low immunogenicity is an obstacle to their use. In this study, we constructed a novel and universal DNA vaccine vector (pSS898) that can be used to build DNA vaccines against duck enteritis virus (DEV) and other viruses that require DNA vaccines to provide protection. This vaccine vector has many advantages, including innate immunogenicity, efficient nuclear trafficking and resistance to attack from nucleases. UL24 and tgB from DEV were chosen as the antigens, and the heat labile enterotoxin B subunit (LTB) from Escherichia coli and the IL-2 gene (DuIL-2) from duck were used as adjuvants for the construction of DNA vaccine plasmids. Ducklings that were orally immunized with S739 (Salmonella Typhimurium Δasd-66 Δcrp-24 Δcya-25) and harboring these DEV DNA vaccines produced strong mucosal and systemic immune responses, and they resisted an otherwise lethal DEV challenge. More importantly, S739 (UL24-LTB) provided 90% protection after a priming-boost immunization. This study shows that our novel and universal DNA vaccine vector can be used efficiently in practical applications and may provide a promising method of orally inoculating ducks with a DEV DNA vaccine delivered by attenuated Salmonella Typhimurium for prevention of DVE.

Evaluation of the Protection Efficacy of a Serotype 1 Marek's Disease Virus-Vectored Bivalent Vaccine Against Infectious Laryngotracheitis and Marek's Disease.

Laryngotracheitis (LT) is a highly contagious respiratory disease of chickens that produces significant economic losses to the poultry industry. Traditionally, LT has been controlled by administration of modified live vaccines. In recent years, the use of recombinant DNA-derived vaccines using turkey herpesvirus (HVT) and fowlpox virus has expanded, as they protect not only against the vector used but also against LT. However, HVT-based vaccines confer limited protection against challenge, with emergent very virulent plus Marek's disease virus (vv+MDV). Serotype 1 vaccines have been proven to be the most efficient against vv+MDV. In particular, deletion of oncogene MEQ from the oncogenic vvMDV strain Md5 (BACδMEQ) resulted in a very efficient vaccine against vv+MDV. In this work, we have developed two recombinant vaccines against MD and LT by using BACδMEQ as a vector that carries either the LT virus (LTV) gene glycoprotein B (gB; BACΔMEQ-gB) or LTV gene glycoprotein J (gJ; BACδMEQ-gJ). We have evaluated the protection that these recombinant vaccines confer against MD and LT challenge when administered alone or in combination. Our results demonstrated that both bivalent vaccines (BACΔMEQ-gB and BACδMEQ-gJ) replicated in chickens and were safe to use in commercial meat-type chickens bearing maternal antibodies against MDV. BACΔMEQ-gB protected as well as a commercial recombinant (r)HVT-LT vaccine against challenge with LTV. However, BACδMEQ-gJ did not protect adequately against LT challenge or increase protection conferred by BACΔMEQ-gB when administered in combination. On the other hand, both BACΔMEQ-gB and BACδMEQ-gJ, administered alone or in combination, protected better against an early challenge with vv+MDV strain 648A than commercial strains of rHVT-LT or CVI988. Our results open a new avenue in the development of recombinant vaccines by using serotype 1 MDV as vectors.

Protective effects of recombinant glycoprotein D based prime boost approach against duck enteritis virus in mice model.

Duck virus enteritis, also known as duck plague, is an acute herpes viral infection of ducks caused by duck enteritis virus (DEV). The method of repeated immunization with a live attenuated vaccine has been used for the prevention and control of duck enteritis virus (DEV). However, the incidence of the disease in vaccinated flocks and latency reactivation are the major constraints in the present vaccination programme. The immunogenicity and protective efficacy afforded by intramuscular inoculation of plasmid DNA encoding DEV glycoprotein D (pCDNA-gD) followed by DEV gD expressed in Saccharomyces cerevisia (rgD) was assessed in a murine model. Compared with mice inoculated with DNA (pCDNA-gD) or protein (rgD) only, mice inoculated with the combination of gD DNA and protein had enhanced ELISA antibody titers to DEV and had accelerated clearance of virus following challenge infection. Furthermore, the highest levels of lymphocyte proliferation response, IL-4, IL-12 and IFN-γ production were induced following priming with the DNA vaccine and boosting with the rgD protein. For instance, the specially designed recombinant DEV vector vaccine would be the best choice to use in ducks. It offers an excellent solution to the low vaccination coverage rate in ducks. We expect that the application of this novel vaccine in the near future will greatly decrease the virus load in the environment and reduce outbreaks of DEV in ducks.

Addition of a UL5 helicase-primase subunit point mutation eliminates bursal-thymic atrophy of Marek's disease virus ∆Meq recombinant virus but reduces vaccinal protection.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus and the causative agent of Marek's disease (MD), characterized by immunosuppression, paralysis, nerve enlargement and induction of T-cell lymphomas in chickens. Despite widespread usage of vaccines since the 1970s to control MD, more virulent field strains of MDV have emerged that overcome vaccinal protection, necessitating the development of new and more protective MD vaccines. The ∆Meq virus, a recombinant Md5 strain MDV lacking the viral oncogene Meq, is one candidate MD vaccine with great potential but unfortunately it also causes bursal-thymic atrophy (BTA) in maternal antibody negative chickens, raising concerns that impede commercial use as a vaccine. Previously, we identified a point mutation within UL5 that reduced in vivo replication in attenuated viruses. We proposed that introduction of the UL5 point mutation into the ∆Meq virus would reduce in vivo replication and eliminate BTA yet potentially retain high protective abilities. In birds, the ∆Meq+UL5 recombinant MDV had reduced replication compared to the original ∆Meq virus, while weights of lymphoid organs indicated that ∆Meq+UL5 did not induce BTA, supporting the hypothesis that reduction of in vivo replication would also abolish BTA. Vaccine trials of the ∆Meq+UL5 virus compared to other ∆Meq-based viruses and commercial vaccines show that, while the ∆Meq+UL5 does provide vaccinal protection, this protection was also reduced compared to the original ∆Meq virus. Therefore, it appears that a very delicate balance is required between levels of replication able to induce high vaccinal protection, yet not so high as to induce BTA.