Systems approach to design multi-epitopic peptide vaccine candidate against fowl adenovirus structural proteins for Gallus gallus domesticus.

Introduction: Fowl adenovirus (FAdV) is a significant pathogen in poultry, causing various diseases such as hepatitis-hydropericardium, inclusion body hepatitis, and gizzard erosion. Different serotypes of FAdV are associated with specific conditions, highlighting the need for targeted prevention strategies. Given the rising prevalence of FAdV-related diseases globally, effective vaccination and biosecurity measures are crucial. In this study, we explore the potential of structural proteins to design a multi-epitope vaccine targeting FAdV. Methods: We employed an in silico approach to design the multi-epitope vaccine. Essential viral structural proteins, including hexon, penton, and fiber protein, were selected as vaccine targets. T-cell and B-cell epitopes binding to MHC-I and MHC-II molecules were predicted using computational methods. Molecular docking studies were conducted to validate the interaction of the multi-epitope vaccine candidate with chicken Toll-like receptors 2 and 5. Results: Our in silico methodology successfully identified potential T-cell and B-cell epitopes within the selected viral structural proteins. Molecular docking studies revealed strong interactions between the multi-epitope vaccine candidate and chicken Toll-like receptors 2 and 5, indicating the structural integrity and immunogenic potential of the designed vaccine. Discussion: The designed multi-epitope vaccine presents a promising approach for combating FAdV infections in chickens. By targeting essential viral structural proteins, the vaccine is expected to induce a robust immunological response. The in silico methodology utilized in this study provides a rapid and cost-effective means of vaccine design, offering insights into potential vaccine candidates before experimental validation. Future studies should focus on in vitro and in vivo evaluations to further assess the efficacy and safety of the proposed vaccine.

Redirect Tropism of Fowl Adenovirus 4 Vector by Modifying Fiber2 with Variable Domain of Heavy-Chain Antibody.

The variable domain of a heavy-chain antibody (VHH) has the potential to be used to redirect the cell tropism of adenoviral vectors. Here, we attempted to establish platforms to simplify the screening of VHHs for their specific targeting function when being incorporated into the fiber of adenovirus. Both fowl adenovirus 4 (FAdV-4) and simian adenovirus 1 (SAdV-1) have two types of fiber, one of which is dispensable for virus propagation and is a proper site for VHH display. An intermediate plasmid, pMD-FAV4Fs, was constructed as the start plasmid for FAdV-4 fiber2 modification. Foldon from phage T4 fibritin, a trigger for trimerization, was employed to bridge the tail/shaft domain of fiber2 and VHHs against human CD16A, a key membrane marker of natural killer (NK) cells. Through one step of restriction-assembly, the modified fiber2 was transferred to the adenoviral plasmid, which was linearized and transfected to packaging cells. Five FAdV-4 viruses carrying the GFP gene were finally rescued and amplified, with three VHHs being displayed. One recombinant virus, FAdV4FC21-EG, could hardly transduce human 293 or Jurkat cells. In contrast, when it was used at a multiplicity of infection of 1000 viral particles per cell, the transduction efficiency reached 51% or 34% for 293 or Jurkat cells expressing exogenous CD16A. Such a strategy of fiber modification was transplanted to the SAdV-1 vector to construct SAdV1FC28H-EG, which moderately transduced primary human NK cells while the parental virus transduced none. Collectively, we reformed the strategy of integrating VHH to fiber and established novel platforms for screening VHHs to construct adenoviral vectors with a specific tropism.

Differential innate immune responses to fowl adenovirus serotype 4 infection in Leghorn male hepatocellular and chicken embryo fibroblast cells.

Fowl adenovirus serotype 4 (FAdV-4) infections result in substantial economic losses in the poultry industry. Recent findings have revealed that FAdV-4 significantly suppresses the host immune response upon infection; however, the specific viral and host factors contributing to this immunomodulatory activity remain poorly characterized. Moreover, diverse cell types exhibit differential immune responses to FAdV-4 infection. To elucidate cell-specific host responses, we performed transcriptomic analysis of FAdV-4 infected leghorn male hepatocellular (LMH) and chicken embryo fibroblast (CEF) cells. Although FAdV-4 replicated more efficiently in LMH cells, it provoked limited interferon-stimulated gene induction. In contrast, FAdV-4 infection triggered robust antiviral responses in CEF cells, including upregulation of cytosolic DNA sensing and interferon-stimulated genes. Knockdown of key cytosolic DNA sensing molecules enhanced FAdV-4 replication in LMH cells while reducing interferon-stimulated gene expression. Our findings reveal cell-specific virus-host interactions that provide insight into FAdV-4 pathogenesis while identifying factors that mediate antiviral immunity against FAdV-4.

Species Fowl aviadenovirus B Consists of a Single Serotype despite Genetic Distance of FAdV-5 Isolates.

Fowl adenoviruses (FAdVs) are infectious agents, mainly of chickens, which cause economic losses to the poultry industry. Only a single serotype, namely FAdV-5, constitutes the species Fowl aviadenovirus B (FAdV-B); however, recently, phylogenetic analyses have identified divergent strains of the species, implicating a more complex scenario and possibly a novel serotype. Therefore, field isolates of the species were collected to investigate the contemporary diversification within FAdV-B, including traditional serotyping. Full genomes of fourteen FAdV-B strains were sequenced and four strains, possessing discriminatory mutations in the antigenic domains, were compared using virus cross-neutralization. Essentially, strains with identical antigenic signatures to that of the first described divergent strain were found in the complete new dataset. While chicken antiserum against FAdV-5 reference strain 340 could not neutralize any of the newly isolated viruses, low homologous/heterologous titer ratios were measured reciprocally. Although they argue against a new serotype, our results indicate the emergence of escape variants in FAdV-B. Charge-influencing amino acid substitutions accounted for only a few mutations between the strains; still, these enabled one-way cross-neutralization only. These findings underline the continued merit of the cross-neutralization test as the gold standard for serotyping, complementary to advancing sequence data, and provide a snapshot of the actual diversity and evolution of species FAdV-B.

FAdV-4 without Fiber-2 Is a Highly Attenuated and Protective Vaccine Candidate.

Hepatitis-hydropericardium syndrome (HHS) caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4) has resulted in huge economic losses to the poultry industry globally. The fiber-2 gene, as a major virulence determiner, is also an important vaccine target against FAdV-4. In this study, we used a CRISPR/Cas9-based homology-dependent recombinant technique to replace the fiber-2 gene with egfp and generate a novel recombinant virus, designated FAdV4-EGFP-rF2. Although FAdV4-EGFP-rF2 showed low replication ability compared to the wild-type FAdV-4 in LMH cells, FAdV4-EGFP-rF2 could effectively replicate in LMH-F2 cells with the expression of Fiber-2. Moreover, FAdV4-EGFP-rF2 was not only highly attenuated in chickens, but also could provide efficient protection against a lethal challenge of FAdV-4. Moreover, FAdV4-EGFP-rF2 without fiber-2 could induce neutralizing antibodies at the same level as FA4-EGFP with fiber-2. These results clearly demonstrate that although fiber-2 affects the viral replication and pathogenesis of FAdV-4, it is not necessary for virus replication and induction of neutralizing antibodies; these findings provide novel insights into the roles of fiber-2 and highlight fiber-2 as an insertion site for generating live-attenuated FAdV-4 vaccines against FAdV-4 and other pathogens. IMPORTANCE Among all serotypes of fowl adenovirus, serotypes FAdV-1, FAdV-4, and FAdV-10 are unique members with two fiber genes (fiber-1 and fiber-2). Recent studies reveal that Fiber-1, not Fiber-2, directly triggers viral infection of FAdV-4, whereas Fiber-2, but not Fiber-1, has been identified as the major virulence determiner and an efficient protective immunogen for subunit vaccines. Here, we replaced fiber-2 with egfp to generate a novel recombinant virus, designated FAdV4-EGFP-rF2. In vitro and in vivo studies on FAdV4-EGFP-rF2 revealed that fiber-2 was not necessary for either virus replication or efficient protection for FAdV-4; these results not only provide a novel live-attenuated vaccine candidate against HHS, but also give new ideas for generating a FAdV-4 based vaccine vector against other pathogens.

Recombinantly Expressed Chimeric Fibers Demonstrate Discrete Type-Specific Neutralizing Epitopes in the Fowl Aviadenovirus E (FAdV-E) Fiber, Promoting the Optimization of FAdV Fiber Subunit Vaccines towards Cross-Protection in vivo.

Vaccines against inclusion body hepatitis in chickens are complicated by the involvement of antigenically diverse fowl adenovirus types. Though immunization with fiber protein confers robust protection, type specificity of fiber antibodies is an obstacle for the desired broad coverage. In this study, we utilized information on multiple linear epitopes predicted in the Fowl Aviadenovirus E (FAdV-E) fiber head (knob) to develop chimeric fibers with an exchange between two serotypes' sequences, each containing proposed epitopes. Two consecutive segments pertaining to amino acid positions 1 to 441 and 442 to 525/523 in the fibers of FAdV-8a and -8b, types of Fowl Aviadenovirus E that cause inclusion body hepatitis, were swapped reciprocally to result in novel chimeras, crecFib-8a/8b and crecFib-8b/8a. crecFib was indistinguishable from monospecific recombinant fibers in its eactivity with different FAdV antisera in Western blotting. However, contrary to the results for monospecific fibers, crecFib induced cross-neutralizing antibodies against both serotypes in chickens. This demonstrates three nonidentical epitopes in the FAdV-E fiber, the conserved epitope detected in Western blotting and at least two epitopes participating in neutralization, being type specific and located opposite residue position 441-442. Furthermore, we supply conformational evidence for a site in the fiber knob with accessibility critical for neutralization. With such an extended neutralization spectrum compared to those of individual fibers, crecFib was anticipated to fulfill and even extend the mechanistic basis of fiber-mediated protection toward bivalent coverage. Accordingly, crecFib, administered as a single-antigen component, protected chickens simultaneously against challenge with FAdV-8a or -8b, demonstrated by up-to-complete resistance to clinical disease, prevention of target organ-related changes, and significant reduction of viral load. IMPORTANCE The control of inclusion body hepatitis, a disease of economic importance for chicken production worldwide, is complicated by an etiology involving multiple divergent fowl adenovirus types. The fiber protein is principally efficacious in inducing neutralizing and protective antibodies in vaccinated chickens; however, it faces limitations due to its intrinsic type specificity for neutralization. In this study, based on an in silico-guided prediction of multiple epitopes in the fowl adenovirus fiber head's loops, we designed chimeric proteins, swapping N- and C-distal fiber portions, each containing putative epitopes, between divergent types FAdV-8a and -8b. In in vitro and in vivo studies, the chimeric fiber displayed extended properties compared to those of individual monotype-specific fibers, allowing the number, distribution, functionality, and conformational bearings of epitopes of the fowl adenovirus fiber to be characterized in more detail. Importantly, the chimeric fiber induced cross-neutralizing antibodies and protective responses in chickens against infections by both serotypes, promoting the advancement of broadly protective subunit vaccination strategies against FAdV.

An inactivated vaccine based on artificial non-pathogenic fowl adenovirus 4 protects chickens against hepatitis-hydropericardium syndrome.

Hepatitis-hydropericardium syndrome (HHS) in birds is mainly caused by virulent fowl adenovirus 4 (FAdV-4). A novel genotype, hypervirulent FAdV-4, emerged in 2015 with a high mortality rate ranging from 30 % to 100 % in chickens. Vaccination is an economically feasible method to control HHS. Although there have been various reports of inactivated vaccines from virulent wild-type FAdV-4 against HHS, biosafety threats of inactivated vaccines from potential pathogenic components have been presented to the poultry industry, and safer vaccines are urgently needed. A non-pathogenic recombinant FAdV-4 strain, designated as rHN20, was generated based on the hypervirulent strain in our previous study. Here, we developed a novel inactivated oil-adjuvanted vaccine derived from rHN20 strain and evaluated its immunogenicity in specific-pathogen-free chickens. Chickens subcutaneously or intramuscularly immunized with the inactivated vaccine produced high titers of neutralizing antibodies and were protected from a lethal dose of virulent wild-type FAdV-4 challenge. Collectively, an inactivated vaccine was developed, which was capable of providing full protection for chickens against HHS, and significantly reduced the potential biosafety threats.

A 10-Year Retrospective Study of Inclusion Body Hepatitis in Meat-Type Chickens in Spain (2011-2021).

A surge in fowl adenovirus (FAdV) causing inclusion body hepatitis (IBH) outbreaks has occurred in several countries in the last two decades. In Spain, a sharp increase in case numbers in broilers and broiler breeder pullets arose since 2011, which prompted the vaccination of breeders in some regions. Our retrospective study of IBH cases in Spain from 2011 to 2021 revealed that most cases were reported in broilers (92.21%) and were caused by serotypes FAdV-8b and -11, while cases in broiler breeder pullets were caused by serotypes FAdV-2, -11, and -8b. Vertical transmission was the main route of infection, although horizontal transmission likely happened in some broiler cases. Despite the inconsistent and heterogeneous use of vaccines among regions and over time, the number of cases mirrored the use of vaccines in the country. While IBH outbreaks were recorded year-long, significantly more cases occurred during the cooler and rainier months. The geographic distribution suggested a widespread incidence of IBH and revealed the importance of a highly integrated system. Our findings contribute to a better understanding of FAdV infection dynamics under field conditions and reiterate the importance of surveillance, serological monitoring of breeders, and vaccination of breeders against circulating serotypes to protect progenies.

Historical Investigation of Fowl Adenovirus Outbreaks in South Korea from 2007 to 2021: A Comprehensive Review.

Fowl adenoviruses (FAdVs) have long been recognized as critical viral pathogens within the poultry industry, associated with severe economic implications worldwide. This specific group of viruses is responsible for a broad spectrum of diseases in birds, and an increasing occurrence of outbreaks was observed in the last ten years. Since their first discovery forty years ago in South Korea, twelve antigenically distinct serotypes of fowl adenoviruses have been described. This comprehensive review covers the history of fowl adenovirus outbreaks in South Korea and updates the current epidemiological landscape of serotype diversity and replacement as well as challenges in developing effective broadly protective vaccines. In addition, transitions in the prevalence of dominant fowl adenovirus serotypes from 2007 to 2021, alongside the history of intervention strategies, are brought into focus. Finally, future aspects are also discussed.

Epidemiology of fowl adenovirus (FAdV) infections in South Korean chickens during 2013-2019 following introduction of FAdV-4 vaccines.

Fowl adenoviruses (FAdV) are important infectious pathogens responsible for causing substantial economic losses to the poultry industry worldwide. One hundred and forty-six FAdV strains were continuously collected and analysed from 2013 to 2019 to understand the epidemiological change and nature of the virus in South Korea from two different standpoints, before and after the release of multiple commercial FAdV-4 vaccines. Phylogenetic analysis of the hexon loop-1 gene sequences showed that 92 strains belonged to FAdV-C (63%), 35 strains to FAdV-E (24%), 18 strains to FAdV-D (12.3%), and one strain to FAdV-A (0.7%), respectively. We provide evidence that the dominant FAdV serotype has recently changed from FAdV-4 to FAdV-8b, as reflected in the proportion of each serotype in field cases in 2019 (18.5% and 77.8%, respectively). The newly emerged FAdV-8b cluster was significantly noticeable compared to the old FAdV clusters, indicating that the development of a vaccine for FAdV-8b may be necessary. Overall, this new insight into FAdV prevalence provides a foundation for strategic control and the development of efficient vaccines against FAdV cases in chickens in South Korea.RESEARCH HIGHLIGHTS The dominant FAdV serotype in South Korea shifted from FAdV-4 to FAdV-8b in 2013-2019.A new cluster of FAdV-8b has emerged in South Korea, indicating the development of new vaccines.

Research Note: Molecular relationship of the fowl adenovirus serotype 4 isolated from the contaminated live vaccine and wild strains isolated in China, 2013-2018.

Since June 2013, hydropericardium-hepatitis syndrome caused by putative novel fowl adenovirus 4 (FAdV-4) infection has spread all over China, leading to great economic losses. Previous study found that the use of attenuated vaccines contaminated with FAdV-4 is likely to be an important cause of such large-scale transmission. Here, we sequenced the whole genome of this strain through the next-generation sequencing and carried out a retrospective analysis of the FAdV-4 strains that have been determined in China recently. Results show the vaccine strain was almost 100% identical with wild virus strains, especially with 4 strains considering the difference of the GA repeat region, further linking the relationship between vaccine contamination and FAdV-4 prevalence in China. Meanwhile, there is no time and regional preference for the emergence of FAdV-4 strains with different molecular characteristics in China, which indicates that there may be multiple routes of transmission of this virus, suggesting that we still need to pay more attention to and formulate correct prevention and control in the future.

An efficient fiber-based ELISA for detection of antibody against fowl adenovirus serotypes 7 and 8.

An outbreak of inclusion body hepatitis caused by fowl adenovirus serotype 8 (FAdV-8) has caused significant economic losses in the poultry industry worldwide. However, a rapid serology test kit specific to FAdV-8 is not available to date. We developed a fiber-based ELISA using the purified GST-fiber of FAdV-8 as coating antigen to measure antibodies against FAdV-8. Specificity analysis showed that our ELISA could react with sera against FAdV-7, -8a, and -8b, but not with sera against the other pathogens tested. Moreover, detection of positive sera with our ELISA had 83% and 94% agreement with an indirect immunofluorescence assay (IFA) and a commercial ELISA from BioChek, respectively. Our ELISA was also effective in the detection of antibodies against FAdV-8 in sera from both experimentally infected and clinically vaccinated chickens. Our FAdV-8 fiber-based ELISA can be a valuable tool to specifically and sensitively detect antibodies against FAdV-7 and/or -8 in infected or vaccinated chickens.

Development and application of a novel ELISA for detecting antibodies against group I fowl adenoviruses.

Since 2015, outbreaks of hepatitis-hydropericardium syndrome (HPS) caused by a novel genotype of fowl adenovirus 4 (FAdV-4) infection have created serious economic losses in China. Given that other serotypes of hypervirulent FAdVs have also been reported in poultry around the world, a common ELISA for all serotypes within the group I fowl adenoviruses (FAdV-I) is urgently needed, especially for clinical epidemic serotypes. In this study, we used high purity and concentration virions of FAdV-4 and developed a common ELISA for detecting antibodies against 12 FAdV-I serotypes. The developed ELISA was able to distinguish between antibodies against FAdV-I, FAdV-III, and other heterologous viruses without any cross-reaction. Furthermore, the ELISA showed higher sensitivity than the FAdV-1-based ELISA to the novel FAdV-4 found in China. Moreover, since there are no commercial vaccines against FAdVs in China, the ELISA was applied to detect sera samples from specific pathogen-free chickens inoculated with inactivated FAdV-1, FAdV-4, and FAdV-8a. The assay showed high sensitivities for all three detected serotypes within FAdV-I. In conclusion, a novel, common ELISA for FAdV-I was developed in this study and could be a powerful tool for seroepidemiological investigations and FAdVs vaccine development.

The fowl adenovirus (Fadv-11) outbreak in Iranian broiler chicken farms: The first full genome characterization and phylogenetic analysis.

Fowl adenoviruses D and E (FAdV-D and E) can cause inclusion body hepatitis (IBH) in commercial chicken flocks. Recently, IBH outbreaks have been increasingly reported in different regions of Iran, particularly in broiler farms. The present study was conducted to perform, for the first time, a complete genome characterization of a FAdV isolate from an IBH outbreak in Iran. Briefly, liver samples were collected from affected broiler flocks and following viral DNA extraction and confirming by PCR technique; one positive sample was selected from an affected flock to conduct a complete genome sequencing. The current FAdV, named "Fowl_Adenovirus_D_isolate_iran/UT-Kiaee_2018", was placed into FAdV-11 serotype (D species). According to the complete genome sequence analysis, UT-Kiaee had high homology with Chinese and Canadian FAdV. The partial sequence of the hexon gene revealed that UT-Kiaee shared 100% identity with previous Iranian FAdVs. The present study was the first to report full genome FAdV in Iran and complete the puzzle of molecular epidemiology of FAdV in Iran through determining the possible origin of Iranian FAdvs, which are the causative agents of recent IBH outbreaks in Iran.

Fowl Adenovirus (FAdV) Recombination with Intertypic Crossovers in Genomes of FAdV-D and FAdV-E, Displaying Hybrid Serological Phenotypes.

After analyzing 27 new genomes from fowl adenovirus (FAdV) field isolates and so-far unsequenced prototypes, we report the first evidence for recombination in FAdVs. Recombination was confined to species FAdV-D and FAdV-E, accommodating the largest number of, and the intraspecies-wise most differentiated, types. The majority of detected events occurred in FAdV-E, involving segments with parental origin of all constitutive types. Together with the diversity of breakpoints, this suggests widespread recombination in this species. With possible constraints through species-specific genes and diversification patterns, the recombinogenic potential of FAdVs attains particular interest for inclusion body hepatitis (IBH), an important disease in chickens, caused by types from the recombination-prone species. Autonomously evolving, recombinant segments were associated with major sites under positive selection, among them the capsid protein hexon and fiber genes, the right-terminal ORFs 19, 25, and the ORF20/20A family. The observed mosaicism in genes indicated as targets of adaptive pressures points toward an immune evasion strategy. Intertypic hexon/fiber-recombinants demonstrated hybrid neutralization profiles, retrospectively explaining reported controversies on reference strains B3-A, T8-A, and X11-A. Furthermore, cross-neutralization supported sequence-based evidence for interdomain recombination in fiber and contributed to a tentatively new type. Overall, our findings challenge the purported uniformity of types responsible for IBH, urging more complete identification strategies for FAdVs. Finally, important consequences arise for in vivo studies investigating cross-protection against IBH.

In silico epitope prediction and immunogenic analysis for penton base epitope-focused vaccine against hydropericardium syndrome in chicken.

Certain strains of fowl adenovirus serotype 4 (FAdV-4) of the family Adenoviridae are recognized to be the causative agents of Hydropericardium Syndrome (HPS) in broiler chicken. Despite the significantly spiking mortality in broilers due to HPS, not much effort has been made to design an effective vaccine against FAdV-4. The combination of immuno- and bioinformatics tools for immunogenic epitope prediction is the most recent concept of vaccine design. It reduces the time and effort required for hunting a potent vaccine candidate and is economical. Previously, we have reported the penton base protein of FAdV-4 to be a candidate for subunit vaccine against HPS. In the present study, we have computationally pre-screened promising B- and T-cell epitopes of the penton base. Multiple methods were employed for linear B-cell epitope identification; BepiPred and five other methods based on physicochemical properties of the amino acids. The penton base was homology modeled by means of Modeller 9.17 and after refinement of the model (by GalaxyRefine web server) ElliPro web tool was used to predict the discontinuous epitopes. NetMHCcons 1.1 and NetMHCIIpan 3.1 servers were used for the likelihood of peptide binding to Major Histocompatibility Complex (MHC) class I & II molecules respectively for T-cell epitope forecast. As a result, we identified the peptide stretch of 1-225  as the most promiscuous B- and T-cell epitope region in penton base Full Length (FL) protein sequence. Escherichia coli based expression vectors were generated containing cloned peptide stretch 1-225 (penton base1-225) and penton base FL gene sequence. The recombinant penton base1-225 and penton base FL proteins were expressed and purified using Escherichia coli-based expression system. Purification yield of penton base1-225 was 3-fold higher compared to penton base FL. These proteins were injected in chickens to determine their competence in protection against HPS. The results showed equal protection level of the two proteins and the commercial inactivated vaccine against FAdV-4 infection. The results suggest the peptide stretch 1-225 of penton base as a valuable candidate for developing an epitope-driven vaccine to combat HPS.

Development of novel subunit vaccine based on truncated fiber protein of egg drop syndrome virus and its immunogenicity in chickens.

Egg-drop syndrome virus (EDSV) is an avian adenovirus that causes markedly decrease in egg production and in the quality of the eggs when it infects chickens. In this report, we engineered truncated fiber protein containing the entire knob domain and part of the shaft region as a vaccine candidate. The protein was obtained in the soluble fraction in Escherichia coli (E. coli), and expression level after nickel-affinity purification was 126 mg/L. By means of multiple characterization methods, it is demonstrated that the recombinant protein retains the native trimeric structure. A single inoculation with the structure-stabilized recombinant protein, even at the lowest dose of 2 µg, stimulated hemagglutination inhibition (HI) antibody responses in chickens, for at least 16 weeks. Neutralizing titers in sera from the protein immunized groups was similar to that of inactivated vaccine immunized group. The lymphocyte proliferation response and cytokine secretion were also induced in immunized SPF chickens. In addition, immunization with the fiber protein also significantly reduced the viral load in the liver. Taken together, these results suggest the truncated fiber protein as an effective single dose, long lasting and rapidly effective vaccine to protect against EDSV.

Pathogenicity of fowl adenovirus (FAdV) serotype 4 strain SDJN in Taizhou geese.

Hydropericardium hepatitis syndrome (HHS) is a fatal disease in chickens, mainly caused by fowl adenovirus serotype 4 (FAdV-4). Since June 2015, HHS has appeared in many provinces in China. The disease has spread from broilers to laying hens, breeders and Cherry Valley ducks, seriously endangering the health of the poultry industry in China. In July 2016, an infectious disease was noticed in a goose farm in Jinan, Shandong Province, China, and hydropericardium was the main finding in post mortem investigations. In the actual study, we isolated a FAdV-4 strain from the livers of naturally-infected goslings and designated it as SDJN. We first evaluated its pathogenicity by inoculating Taizhou geese at 10, 20, and 30 days of age with 10-7.15EID50/0.2 ml doses of the SDJN strain in 1 ml allantoic fluid via subcutaneous injection or oral infection. Clinical signs and pericardial effusion appeared in geese infected subcutaneously at 10 days of age, whereas 20- and 30-day-old geese were not susceptible to FAdV-4. The results of real-time PCR showed that the replication ability of FAdV-4 in geese correlated with the age. Furthermore, results from clinical chemistry showed that FAdV-4 damaged the liver and kidney in geese and the results paralleled viral load and gross lesions. Consequently, FAdV-4 was pathogenic in geese, and the pathogenicity was related to age and mode of infection. This study is the first experimental infection of FAdV-4 in geese, which will provide a basis for further understanding of the disease. RESEARCH HIGHLIGHTS Pathogenicity tests with a FAdV-4 were conducted in geese, which included data on clinical signs, gross pathology, histopathology, clinical chemistry and viral load. FAdV-4 could replicate in geese and HHS was successfully induced. Pathogenicity of FAdV-4 in geese was related to the age and routes of infection.

Vaccination with FAdV-8a induces protection against inclusion body hepatitis caused by homologous and heterologous strains.

Fowl aviadenoviruses (FAdV) are important avian pathogens, responsible for several poultry diseases prevalent worldwide, including inclusion body hepatitis (IBH). FAdV intraspecies cross-protection has been clearly demonstrated, but there is little evidence that any interspecies cross-protection exists. The present study aimed to assess the inter- and intraspecies protection between three FAdV field isolates (FAdV-8a, FAdV-8b, FAdV-11) identified in association with severe IBH outbreaks. Inocula prepared using inactivated plaque-purified virus with adjuvant Montanide™ ISA 71VG, were injected intramuscularly into 3-week-old SPF chickens. At 6-weeks of age, the birds were challenged with 106 TCID50 of homologous or heterologous virus intraperitoneally, and full post mortem examination performed at 4 days post-challenge. Various tissues were examined for gross and histological lesions and assessed for the presence of virus by PCR-HRM. All homologous-type vaccine/challenge groups exhibited protection against IBH lesions with no virus detected in the tissues. Unvaccinated groups challenged with virus showed evidence of FAdV-induced lesions; however, FAdV-8a demonstrated lower pathogenicity compared with FAdV-8b and FAdV-11. In the heterologous-type vaccine/challenge groups, FAdV-8a vaccine was shown to protect against challenge with both FAdV-8b and FAdV-11. FAdV-8a and 8b belong to species E and were therefore anticipated to cross-protect. However, FAdV-11 belongs to species D and therefore cross-protection by FAdV-8a was an uncharacteristic and unique finding of this study. Further research is required to disseminate the molecular basis for the interspecies cross-protection between FAdV-8a and FAdV-11. Nonetheless, the FAdV-8a isolate was shown to have substantial potential as a vaccine candidate in countries where FAdV-8a, 8b or 11 are prevalent.

A novel monoclonal antibodies-based sandwich ELISA for detection of serotype 4 fowl adenovirus.

As a major causative agent for hepatitis-hydropericardium syndrome (HPS) in chickens, serotype 4 fowl adenovirus (FAdV-4) has caused huge economic losses in the poultry industry globally. However, there is no commercial diagnostic test for FAdV-4 antigens. To generate a rapid approach for specific detection of FAdV-4, a monoclonal antibodies (mAbs)-based sandwich ELISA was developed. In this ELISA, a purified mAb 4A3 and a HRP-labelled mAb 3C2 specific to the fiber-2 of FAdV-4 were used as the capture antibody and detection antibody respectively. Specificity assay revealed the ELISA only reacted with FAdV-4, not with other avian viruses tested. Sensitivity assay showed the limit of detection of the ELISA was 1000 TCID50/ml and 12.5 ng/ml for the FAdV-4 and the purified GST-Fiber2 protein respectively. Moreover, the ELISA could be efficiently applied in detecting the FAdV-4 in tissue samples from a clinically-diseased chicken flock. All these data demonstrated that the ELISA developed here provides a promising tool for rapid and efficient diagnosis of clinical infection with FAdV-4.