Avian Adenovirus Research Articles

Oncolytic viruses in cancer therapy

Oncolytic virotherapy is a promising form of gene therapy for cancer, employing nature’s own agents to find and destroy malignant cells. The purpose of this review is to provide an introduction to this very topical field of research and to point out some of the current observations, insights and ideas circulating in the literature. We have strived to acknowledge as many different oncolytic viruses as possible to give a broader picture of targeting cancer using viruses. Some of the newest additions to the panel of oncolytic viruses include the avian adenovirus, foamy virus, myxoma virus, yaba-like disease virus, echovirus type 1, bovine herpesvirus 4, Saimiri virus, feline panleukopenia virus, Sendai virus and the non-human coronaviruses. Although promising, virotherapy still faces many obstacles that need to be addressed, including the emergence of virus-resistant tumor cells.

Genetic Characterization, Pathogenicity, and Protection Studies with an Avian Adenovirus Isolate Associated with Inclusion Body Hepatitis

An avian adenovirus (AAV) was isolated from liver samples of two 2-wk-old broiler-breeder flocks obtained from grandparents vaccinated at 10 and 17 wks of age with an autogenous inactivated vaccine containing the European AAV 8 (8565 strain) and 11 (1047 strain) serotypes (AAV8/11 vaccine). Affected broiler-breeders exhibited clinical signs and macroscopic and microscopic lesions associated with inclusion body hepatitis (IBH). The isolated adenovirus, identified as Stanford, was molecularly characterized as European serotype 9. The pathogenicity of the Stanford strain was confirmed after inoculation of specific-pathogen-free (SPF) chickens at 1-7 days of age, causing 100% and 20% mortality, respectively. The level of protection against IBH was evaluated in two broiler-breeder progenies from AAV 8/11-vaccinated grandparent flocks and a commercial broiler flock by challenge at 1 or 7 days of age with the AAV 8 and 11 serotypes and/or the Stanford strain. The broiler-breeder progenies and the commercial broiler flock exhibited protection against IBH after challenge. No significant differences in mean body weights were observed at 3 wk of age in any of the evaluated groups. We conclude that broiler-breeder progenies from 30- to 50-wk-old grandparents vaccinated with the AAV 8/11 vaccine were adequately protected against challenge with the AAV 8 and 11 serotypes and the Stanford strain.

Genetic Characterization, Pathogenicity, and Protection Studies with an Avian Adenovirus Isolate Associated with Inclusion Body Hepatitis

Genetic Characterization, Pathogenicity, and Protection Studies with an Avian Adenovirus Isolate Associated with Inclusion Body Hepatitis

Effects of Polyether Ionophores on the Protective Immune Responses of Broiler Chickens against Angara Disease and Newcastle Disease Viruses

Immunization against Angara disease virus (ADV), a serotype 4 avian adenovirus, and Newcastle disease virus (NDV), an avian paramyxovirus serotype 1, is the mainstay of a broiler vaccination programme, while polyether ionophores usually form an essential component of a broiler medication programme in most parts of India and Pakistan. The role of polyether ionophores in the protective immune responses of broiler chickens vaccinated and challenged with ADV and NDV was investigated. A total of 1600 birds were divided into eight groups of 200 birds each. First four groups were vaccinated against NDV and ADV, while the remaining four served as unvaccinated controls. The first 3 groups of birds were administered salinomycin, monensin and cyclophosphamide (CYP), respectively. The last group served as an untreated control. The same treatment schedule was also followed for the next four unvaccinated groups. The post-vaccination and post-challenge serological responses to NDV and ADV, body and lymphoid organ weight gains, post-challenge survival rate and detection of NDV and ADV in the tissues of infected birds were evaluated. Birds administered salinomycin showed a significant stimulation of protective immune responses against both NDV and ADV as compared to the untreated and CYP-treated birds. Monensin also enhanced the protective immune responses against both viruses but the effect was not statistically significant. Thus, it is concluded that monensin and salinomycin augment the anti-NDV and anti-ADV immune responses in broiler chickens, which supports their use in poultry flocks.

Nanoporous crystals of chicken embryo lethal orphan (CELO) adenovirus major coat protein, hexon

CELO (chicken embryo lethal orphan) virus is an avian adenovirus that is being developed as a gene transfer vector. Its trimeric major coat protein (942 residues, 106,709 Da) has 42% sequence identity to human adenovirus type 2 (AdH2) hexon and 45% to AdH5 hexon. For structural studies, the growth of CELO virus has been optimized, and its hexon purified and crystallized. The hexon crystals, the first non-human example, diffract to 3.9 Å resolution. Molecular replacement using the AdH5 model was used to identify the location of the CELO hexon within the unit cell. There is one hexon monomer in the asymmetric unit of the trigonal space group P321 ( a = b = 157.8 Å, c = 114.2 Å, γ = 120°) and the solvent content is 67.8%. The hexons pack in a hexagonal honeycomb so that large ∼100 Å diameter channels run through the entire crystal. This remarkable property of the crystals lends itself to their exploitation as a nanomaterial. Structural studies on CELO will elucidate the differences between avian and human adenoviruses and contribute to a better understanding of adenoviruses with non-human hosts.

HEMATOLOGY, SERUM CHEMISTRY, AND SEROLOGY OF GALÁPAGOS PENGUINS (SPHENISCUS MENDICULUS) IN THE GALÁPAGOS ISLANDS, ECUADOR

The Galápagos penguin (Spheniscus mendiculus) is an endangered species endemic to the Galápagos Islands, Ecuador. In 2003 and 2004, 195 penguins from 13 colonies on the islands of Isabela and Fernandina in the Galápagos archipelago were examined. Genetic sexing of 157 penguins revealed 62 females and 95 males. Hematology consisted of packed cell volume (n = 134), white blood cell differentials (n = 83), and hemoparasite blood smear evaluation (n = 114). Microfilariae were detected in 22% (25/114) of the blood smears. Female penguins had significantly higher eosinophil counts than males. Serum chemistry on 83 penguins revealed no significant differences between males and females. Birds were seronegative to avian paramyxovirus type 1-3, avian influenza virus, infectious bursal disease virus, Marek's disease virus (herpes), reovirus, avian encephalomyelitis virus, and avian adenovirus type 1 and 2 (n = 75), as well as to West Nile virus (n = 87), and Venezuelan, western and eastern equine encephalitis viruses (n = 26). Seventy-five of 84 (89%) penguins had antibodies to Chlamydophila psittaci but chlamydial DNA was not detected via polymerase chain reaction in samples from 30 birds.

Crystallization of the C-terminal head domain of the avian adenovirus CELO long fibre

Avian adenovirus CELO contains two different fibres: fibre 1, the long fibre, and fibre 2, the short fibre. The short fibre is responsible for binding to an unknown avian receptor and is essential for infection of birds. The long fibre is not essential, but is known to bind the coxsackievirus and adenovirus receptor protein. Both trimeric fibres are attached to the same penton base, of which each icosahedral virus contains 12 copies. The short fibre extends straight outwards, while the long fibre emerges at an angle. The carboxy-terminal amino acids 579-793 of the avian adenovirus long fibre have been expressed with an amino-terminal hexahistidine tag and the expressed trimeric protein has been purified by nickel-affinity chromatography and crystallized. Crystals were grown at low pH using PEG 10,000 as precipitant and belonged to space group C2. The crystals diffracted rotating-anode Cu Kalpha radiation to at least 1.9 angstroms resolution and a complete data set was collected from a single crystal to 2.2 angstroms resolution. Unit-cell parameters were a = 216.5, b = 59.2, c = 57.5 angstroms, beta = 101.3 degrees, suggesting one trimer per asymmetric unit and a solvent content of 46%. The long fibre head does not have significant sequence homology to any other protein of known structure and molecular-replacement attempts with known fibre-head structures were unsuccessful. However, a map calculated using SIRAS phasing shows a clear trimer with a shape similar to known adenovirus fibre-head structures. Structure solution is in progress.

A broadly applicable method to characterize large DNA viruses and adenoviruses based on the DNA polymerase gene

BackgroundMany viral pathogens are poorly characterized, are difficult to culture or reagents are lacking for confirmatory diagnoses. We have developed and tested a robust assay for detecting and characterizing large DNA viruses and adenoviruses. The assay is based on the use of degenerate PCR to target a gene common to these viruses, the DNA polymerase, and sequencing the products.ResultsWe evaluated our method by applying it to fowl adenovirus isolates, catfish herpesvirus isolates, and largemouth bass ranavirus (iridovirus) from cell culture and lymphocystis disease virus (iridovirus) and avian poxvirus from tissue. All viruses with the exception of avian poxvirus produced the expected product. After optimization of extraction procedures, and after designing and applying an additional primer we were able to produce polymerase gene product from the avian poxvirus genome. The sequence data that we obtained demonstrated the simplicity and potential of the method for routine use in characterizing large DNA viruses. The adenovirus samples were demonstrated to represent 2 types of fowl adenovirus, fowl adenovirus 1 and an uncharacterized avian adenovirus most similar to fowl adenovirus 9. The herpesvirus isolate from blue catfish was shown to be similar to channel catfish virus (Ictalurid herpesvirus 1). The case isolate of largemouth bass ranavirus was shown to exactly match the type specimen and both were similar to tiger frog virus and frog virus 3. The lymphocystis disease virus isolate from largemouth bass was shown to be related but distinct from the two previously characterized lymphocystis disease virus isolates suggesting that it may represent a distinct lymphocystis disease virus species.ConclusionThe method developed is rapid and broadly applicable to cell culture isolates and infected tissues. Targeting a specific gene for in the large DNA viruses and adenoviruses provide a common reference for grouping the newly identified viruses according to relatedness to sequences of reference viruses and the submission of the sequence data to GenBank will build the database to make the BLAST analysis a valuable resource readily accessible by most diagnostic laboratories. We demonstrated the utility of this assay on viruses that infect fish and birds. These hosts are phylogenetically distant from mammals yet, sequence data suggests that the assay would work equally as well on mammalian counterparts of these groups of viruses. Furthermore, we demonstrated that obtaining genetic information on routine diagnostic samples has great potential for revealing new virus strains and suggesting the presence of new species.

Evaluation of the health of free-ranging greater rheas (Rhea americana) in Argentina

The health of 22 free-ranging adult rheas (Rhea americana) examined and sampled during a translocation/reintroduction project and six juvenile rheas kept in semicaptivity was investigated, and details of their haematology...

HEMATOLOGY, PLASMA CHEMISTRY, AND SEROLOGY OF THE FLIGHTLESS CORMORANT (PHALACROCORAX HARRISI) IN THE GALÁPAGOS ISLANDS, ECUADOR

The flightless cormorant (Phalacrocorax harrisi) is an endemic species of the Galápagos Islands, Ecuador. Health studies of the species have not previously been conducted. In August 2003, baseline samples were collected from flightless cormorant colonies on the islands of Isabela and Fernandina. Seventy-six birds, from nestlings to adults, were evaluated. Genetic sexing of 70 cormorants revealed 37 females and 33 males. Hematology assessment consisted of packed cell volume (n=19), leukograms (n=69), and blood smear evaluation (n=69). Microscopic evaluation of blood smears revealed microfilaria in 33% (23/69) of the cormorants. Plasma chemistries were performed on 46 cormorants. There was no significant difference in chemistry values or complete blood counts between male and female cormorants or between age groups. Based on a serologic survey to assess exposure to avian pathogens, birds (n=69) were seronegative for West Nile virus, avian paramyxovirus type 1 (Newcastle disease virus), avian paramyxovirus types 2 and 3, avian influenza, infectious bursal disease, infectious bronchitis, Marek's disease (herpes), reovirus, avian encephalomyelitis, and avian adenovirus type 2. Antibodies to avian adenovirus type 1 and Chlamydophila psittaci were found in 31% (21/68) and 11% (7/65) of flightless cormorants respectively. Chlamydophila psittaci was detected via polymerase chain reaction in 6% (2/33) of the cormorants. The overall negative serologic findings of this research suggest that the flightless cormorant is an immunologically naïve species, which may have a reduced capacity to cope with the introduction of novel pathogens.

Comparative study of hydropericardium hepatitis syndrome vaccine in poultry

Two commercial types of Hydro pericardium Hepatitis Syndrome (HHS) oily inactivated vaccines were evaluated. The first was (Angavac) French in origin (Merial Co.) & the 2nd is (Nobilis Hepatitis) Holland in origin (Intervet Co.). A unified program of vaccination had been used for both vaccines. One & ten days old chicks were vaccinated by subcutaneous & intramuscular route of injection with both vaccines in each age separately . Serum antibodies titer weekly measured by using Indirect haemagglutination test (IHA) & agar gel diffusion test. Challenge test at 4 week of age was done by virulent avian adeno virus serotype – 4 (VAAV-4) as well as virulent field isolate was used as challenge virus. The result showed highest antibodies titer at week age. Angavac vaccine gave highest titer than Nobilis Hepatitis vaccine & IHA was more sensitive than AGDT. All groups vaccinated with Angavac gave 100% protection in challenge test with (VAAV-4) at 28 days. While in Holland vaccine the protection ratio was 60-80% only, but the ratio of protection of both vaccines did not exceed than 40-50% when the field isolate was used as challenge virus.

Partial Characterization of an Adenovirus-Like Virus Isolated from Broiler Chickens with Transmissible Viral Proventriculitis

Transmissible viral proventriculitis (TVP) was experimentally reproduced in specific-pathogen-free chickens using a homogenate of proventricular tissue obtained from TVP-affected commercial broiler chickens. Thin-section electron microscopy revealed intranuclear, approximately 70-nanometer (nm), adenovirus-like viruses (AdLV) within proventricular lesions. The AdLV, designated AdLV (R11/3), could not be propagated using various avian and mammalian cell cultures or by inoculation of embryonated chicken eggs by yolk, allantoic, or chorioallantoic membrane routes. However, AdLV (R11/3) was successfully propagated by amniotic inoculation of embryonated chicken eggs, with detection of the virus in proventriculi and intestinal contents of hatched 2-day-old chicks (8 days postinoculation). Virus propagation was evident in in ovo-inoculated chicks by (1) gross and microscopic lesions in proventriculi consistent with TVP, (2) immunohistochemical localization of AdLV (R11/3) antigens in proventricular epithelium, (3) thin-section electron microscopic detection of intranuclear, approximately 70-nm AdLVs within proventricular epithelium, and (4) negative-stain electron microscopic detection of extracellular, approximately 70-nm AdLVs in intestinal contents. Indirect immunofluorescence and polymerase chain reaction procedures that specifically recognize groups I, II, and III avian adenoviruses failed to recognize AdLV (R11/3). The findings suggest an etiologic role for AdLV (R11/3) in TVP and indicate that this virus is distinct from known avian adenoviruses.

Deletion of open reading frames 9, 10 and 11 from the avian adenovirus CELO genome: effect on biodistribution and humoral responses

In this study, the in vivo effect of the 3.6 kbp deletion of the three open reading frames (ORF) 9, 10 and 11 found at the right end of the CELO genome was examined. Groups of chickens were inoculated oronasally with 10(5)-10(7) p.f.u. per animal of wild-type virus and two recombinant CELO strains (rCELO) expressing luciferase and secreted alkaline phosphatase (SEAP). The tissue biodistribution, assessed by PCR, was similar for both wild-type and recombinant viruses. The infectious viral particle titre was determined by a p.f.u. counting method and the antibody responses to the CELO vector and the SEAP antigen were evaluated by ELISA. Infectious particle titres in tissues from chickens inoculated with the wild-type CELO virus increased up to 6 days post-inoculation, and declined until 11 days while titres in organs from chickens inoculated with the rCELO strain were low and only detectable at 4 days post-inoculation. Moreover, although anti-CELO antibody levels were three times lower in sera from chickens inoculated with rCELO, antibodies directed to the heterologous SEAP antigen were detected. Based on these results, no differences in tropism were observed, but the level of production of viral particles and the humoral responses appeared to decrease. Viruses replicate less efficiently with a deletion performed at the right end of the CELO genome. Nevertheless, the presence of antibodies directed to heterologous antigens makes the CELO virus an advantageous candidate for avian vaccination.

Phylogenetic analysis of the hexon loop 1 region of an adenovirus from psittacine birds supports the existence of a new psittacine adenovirus (PsAdV)

Adenovirus infections in psittacine birds have been well known. Most of these infections were caused by fowl adenoviruses (FAdV). In this study, liver samples showing typical histological signs of an adenovirus infection were collected from Poicephalus spp. with acute disease. A PCR amplifying the variable loop 1 region of the hexon gene was developed using primers located in two conserved pedestal regions. A PCR product of approximately 590 bp in size was amplified and sequenced. The sequence obtained grouped outside of the FAdV reference strains of the 12 serotypes as well as egg drop syndrome virus and turkey adenovirus 3 indicating that a new avian adenovirus was detected. In comparison to the FAdV reference strains, the percentage of identical nucleotides ranged between 60.3 and 67.0 and that of identical amino acids (aa) between 51.3 and 61.0. Furthermore, 37 unique aa exchanges were observed; out of these, 27 are located in the 4 hypervariable regions of loop 1, which encode the serotype-specific epitopes. The g/c content, the isoelectric point and the charge of the amplified fragment, however, are in the range as those of group I avian adenoviruses. It was proposed, therefore, to designate this new adenovirus as psittacine adenovirus (PsAdV).

396. Identification and Characterization of Novel AAV Isolates

AAV is a known contaminant of adenovirus preparations including stocks of human, simian, bovine, avian, and canine adenoviruses. While other viruses, such as HSV and vaccinia, can function as helper viruses for AAV replication in vitro, AAV has not been identified as a contaminant of these viral preparations. In order to to identify helper viruses that allow propagation of AAV and to identify and characterize novel AAV isolates, we screened about 100 viral stocks, supplied by the American Type Culture Collection (ATCC) for the presence of AAV DNA using a PCR based assay. These samples included adenovirdae, herpesviridae, retroviridae, coronaviridae and orthomyxoviridae. Thirty-three percent of the adenovirus stocks screened were contaminated with AAV with the highest percentage (79%) found in human and non-human primate adenovirus samples. We were unable to detect AAV in any other virus family tested, suggesting that adenoviruses are the preferred helper virus. We cloned and sequenced the genomes of AAVs found in ten simian adenovirus isolates. These novel AAV isolates displayed at least 98% homology in the capsid ORF to either AAV1, AAV6, or to each other. The majority of divergent amino acids between the novel AAVs and AAV6 are located on the surface of the virus surrounding the 3-fold axis of symmetry, in an area of the capsid that has been associated with receptor binding. Despite their high homology to AAV-6, we observed a distinct cell tropism of the novel AAV isolates and differences in sensitivity to lectin competition, suggesting that these novel isolates may utilize a distinct entry pathway. These novel AAVs might prove useful for gene therapy applications and we are currently investigating the use of the novel AAV isolates in in-vivo gene transfer.

Avian adenovirus vector CELO-TK displays anticancer activity in human cancer cells and suppresses established murine melanoma tumors

Avian adenovirus CELO is a novel adenovirus vector system with the advantages of efficient production, high virion stability, and the absence of crossreactivity with Ad5-neutralizing antibodies. In this study, we evaluated the anticancer efficacy of a CELO vector encoding the herpes simplex virus type 1 thymidine kinase, a prodrug-activating therapeutic gene. Vectors carrying the gene for HSV-tk or EGFP under the control of the HCMV promoter in place of the "nonessential" region of the CELO genome were constructed. Anticancer activity of the CELO-TK vector was studied in vitro, in human and murine tumor cells in cell culture, and in vivo, in established subcutaneous murine B16 melanoma tumors in C57BL/6 mice. The CELO-TK vector mediated delivery of functional HSV-tk to tumor cell lines in cell culture. Comparison of the CELO-TK vector to a first-generation human adenovirus type 5 vector Ad5-TK in cultured H1299 cells showed equal levels of functional activity at increasing multiplicities of infection with CELO-based vector. CELO vectors allowed for transduction and expression of EGFP and HSV-tk genes in subcutaneous melanoma tumors in C57BL/6 mice. Intratumoral injections of CELO-TK followed by ganciclovir administration resulted in suppression of tumor growth and significantly increased the median of survival. The results of the study demonstrated the efficacy of CELO vector as a vehicle for the delivery of prodrug-activating genes such as HSV-tk to tumor cells in vitro and in vivo.

The CELO adenovirus Gam1 protein enhances transient and stable recombinant protein expression in Chinese hamster ovary cells

The Gam1 protein of the avian CELO adenovirus activates transcription through inhibition of histone deacetylase 1 (HDAC1). We investigated the effect of Gam1 on both transient and stable transgene expression in Chinese hamster ovary (CHO) cells, one of the most commonly used mammalian hosts for the large-scale production of recombinant proteins. Transient expression of Gam1 increased reporter protein levels up to 4-fold in suspension cultures of CHO DG44 cells co-transfected with a reporter gene and up to 20-fold in recombinant CHO DG44-derived cell lines. The highest levels of activation were observed when the transgene was under the control of the human cytomegalovirus (HCMV) immediate early promoter/enhancer. Increases in recombinant protein expression in the presence of Gam1 were not accompanied by an enhancement of cell growth or viability. We conclude that Gam1 may serve as a useful genetic tool for increasing recombinant protein expression in CHO DG44 cells.

Sequence analysis, viral rescue from infectious clones and generation of recombinant virions of the avian adeno-associated virus

Aiming at the generation of a viral-vectored system for gene delivery and vaccination in poultry, the entire genomes of the VR-865 and DA-1 strains of the avian adeno-associated virus have been cloned and sequenced. Sequence analysis of the clones showed that the genomic distribution of the structural and non-structural protein-coding genes of these viruses is conserved and in agreement with what has been previously described for the primate adeno-associated viruses. Amino acid differences between the avian adeno-associated viruses and the primate adeno-associated viruses are more evident in the genes that code for the non-structural (Rep) proteins of the virus, while the Cap region amino acid sequence was found to be more conserved. Since all the regulatory and coding sequences of the virus were present in the plasmids obtained, complete infectious viral particles were rescued from these clones, and these rescued viral populations were amplified by co-infecting primary embryo liver cells with the rescued virus and the CELO strain of the avian adenovirus type 1. As a proof of concept of the validity of this system for the purpose of gene delivery, recombinant viruses encoding for the LacZ gene as a reporter system were also generated. These recombinant viruses were used to express beta galactosidase activity in primary chicken embryo cell cultures.

Development of a multiplex PCR for detection of avian adenovirus, avian reovirus, infectious bursal disease virus, and chicken anemia virus

A multiplex polymerase chain reaction (mPCR) was developed and optimized for the simultaneous detection and differentiation of avian reovirus (ARV), avian adenovirus group I (AAV-I), infectious bursal disease virus (IBDV), and chicken anemia virus (CAV). Four sets of specific oligonucleotide primers were used in this test for ARV, AAV-I, IBDV, and CAV. The mPCR DNA products were visualized by gel electrophoresis and consisted of fragments of 365 bp for IBDV, 421 bp for AAV-I, 532 bp for ARV, and 676 bp for CAV. The mPCR assay developed in this study was found to be sensitive and specific. Detection of PCR-amplified DNA products was 100 pg for both CAV and IBDV, and 10 pg for both ARV and AAV-I and this mPCR did not amplify nucleic acids from the other avian pathogens tested. The mPCR demonstrated similar sensitivity in tests using experimental fecal cloacal swab specimens that were spiked with ARV, AAV-1, IBDV, and CAV, and taken from specific pathogen free (SPF) chickens. This mPCR detected and differentiated various combinations of RNA/DNA templates from ARV, AAV-I, CAV, and IBDV without reduction of amplification from feces.

Avian adenovirus CELO recombinants expressing VP2 of infectious bursal disease virus induce protection against bursal disease in chickens

To develop a CELO virus vector that can induce protection against infectious bursal disease, CELO viruses expressing the host-protective antigen VP2 of infectious bursal disease virus (IBDV) were constructed. In the engineered recombinants, the VP2 gene (the 441-first codons of the IBDA polyprotein) was placed under the control of the CMV promoter. Two positions in the CELO genome were chosen to insert the VP2 expression cassette. The recombinants were found apathogenic, when inoculated by different routes and even at high doses (up to 10 8 per animal). Chickens vaccinated oro-nasally with these different recombinants and challenged with very virulent IBDV were found to be poorly protected. In contrast, when inoculated with one or two (subcutaneous or intradermic) injections of CELOa-VP2, the chickens showed no clinical signs and no mortality after challenge. In the vaccinated chickens, the titers of neutralization antibody reached 7–9 values, showing that protection could be explained by the induction of a sufficient humoral response. After challenge, the weight ratio Bursa of Fabricius/body was about 2.5‰, a value similar to that obtained with the commercial Bur706 vaccine. However, histological lesions in the Bursa of Fabricius were observed, showing that a complete protection was not totally achieved. Contact transmission was evidenced. Protection was also obtained when inoculation of CELOa-VP2 was carried out in ovo. Prime-boost strategies were also tested with the CELOa-VP2 vector used in association with the purified VP2 antigen, or DNA encoding VP2 or a CELO vector expressing chicken myeloid growth factor (cMGF). None of these regimens were shown to substantially increase the level of protection when compared to double CELOa-VP2 inoculations. These results indicate that CELO-based vectors are useful to safely induce a strong protective immunity against vvIBDV in chickens.