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.

Efficacy, immunogenicity, and virus shedding in broiler chickens inoculated with live attenuated fowl adenovirus serotype 8b propagated a bioreactor.

Background: Fowl adenovirus (FAdV) 8b causes huge economic losses in the poultry industry worldwide. Attenuated FAdV 8b could be useful in preventing FAdV infections globally and scale-up obstacles could be solved by bioreactor technology. Aim: This study was carried out to attenuate the FAdV 8b isolate, propagate it in a bioreactor, molecularly characterize the passage isolates, and determine the immunogenicity, efficacy, and shedding of the virus of chickens. Methods: FAdV serotype 8b (UPM11142) isolate was passaged on chicken embryo liver (CEL) cells until attenuation and propagated in a bioreactor (UPM11142P20B1). Hexon and fiber genes of the isolates were sequenced and analyzed. UPM11142P20B1 was administered to 116-day-old broiler chickens divided into four groups, A (control), B (non-booster), C (booster with UPM11142P20B1), and D (booster with inactivated UPM11142P5B1). Eight chickens from each group were challenged. Body weight (BW) and liver weight (LW), liver: BW ratio (LBR), FAdV antibody titer, T lymphocyte sub-populations in the liver, spleen and thymus; and challenge virus load in the liver and shedding in cloaca were measured at weekly intervals. Results: The isolate caused typical cytopathic effects on CEL cells typical of FAdV. Novel molecular changes in the genes occurred which could be markers for FAdV 8b attenuation. BW, LW, and LBR were similar among groups throughout the trial but the uninoculated control-challenged group (UCC) had significantly higher LBR than the inoculated and challenged groups at 35 dpi. Non-booster group had higher FAdV antibodies at all time points than the uninoculated control group (UCG); and the challenged booster groups had higher titer at 35 dpi than UCC. T lymphocytes increased at different time-points in the liver of inoculated chickens, and in the spleen and thymus as well, and was higher in the organs of inoculated challenged groups than the UCC. There was a significantly higher challenge virus load in the liver and cloaca of UCC chickens than in the non-booster chickens. Conclusion: UPM11142P20B1 was safe, efficacious, significantly reduced shedding, and is recommended as a candidate vaccine in the prevention and control of FAdV 8b infections in broiler chickens.

Genomic characterization and pathogenicity of a novel fowl adenovirus serotype 11 isolated from chickens with inclusion body hepatitis in China.

Fowl adenovirus serotype 11 (FAdV-11) is one of the primary causative agents of inclusion body hepatitis (IBH), which causes substantial economic losses in the world poultry industry. In this study, we characterized the genome of the fowl adenovirus serotype 11 (FAdV-11) isolate FJSW/2021. The full genome of FJSW/2021 was 44, 154 base pairs (bp) in length and had a similar organization to that of previously reported FAdV-11 isolates. Notably, compared with those of other reported FAdV-11 strains, the preterminal protein (pTP) of FAdV-11 FJSW/2021 has six amino acid (aa) insertions (S-L-R-I-I-C) between 470 and 475 and one aa mutation of L476F; moreover, the tandem repeat (TR) regions of TR1 and TR2 were 33 bp (1 repeat) and 1,080 bp (8 repeats) shorter than those of the Canadian nonpathogenic isolate ON NP2, respectively. The pathogenicity of FJSW/2021 was studied in 10-day-old specific pathogen-free chicken embryos following allantoic cavity inoculation and in 1-day-old, 1-wk-old and 2-wk-old SPF chickens following intramuscular inoculation with 107 TCID50 of the virus. The results showed that FJSW/2021 can induce typical severe IBH in chicks less than 2 wk old. These findings highlighted the genetic differences between the pathogenic and non-pathogenic FAdV-11 isolates. The data will provide guidance for identifying the virulence factors of FAdV-11 strains. The animal challenge model developed in our study will allow precise evaluation of the efficacy of potential FAdV-11 vaccine candidates.

Development of an ELISA for the detection of fowl adenovirus serotype -4 utilizing fiber protein.

Hydropericardium syndrome (HPS), caused by the Fowl adenovirus 4 (FAdV-4) has led to significant financial losses for the poultry industry globally, including Pakistan over the past few years. Conventional serological methods are time consuming, laborious and less sensitive therefore, a rapid and sensitive ELISA kit is required for the reliable detection of FAdV-4 infection. In the current research, fiber proteins (1 &2) of FAdV-4 were successfully expressed in Escherichia coli and purified using metal affinity chromatography. Using these proteins as antigens, an indirect ELISA for detecting FAdV-4 infection was developed. The developed ELISA showed superior performances upon comparison with Serum neutralization test (SNT). This ELISA also showed reliable detection of FAdV specific antibodies in experimentally infected and vaccinated chickens. This assay produced good correlation on the samples collected from the field with SNT and found essential for large scale serology of the FAdV. No cross reactivity was observed in the ELISA following the testing of the serum samples of different other avian pathogens which showed that this ELISA is specific in detecting the FAdV infection. In conclusion, the developed Fiber protein ELISA is highly sensitive and specific in the detecting the FAdV infection and can be utilized for large scale sero-epidemiology of the disease.

Genome analysis of a novel avian atadenovirus reveals a possible horizontal gene transfer.

We report the discovery and characterization of a novel adenovirus, Zoothera dauma adenovirus (ZdAdV), from a wild bird species, Zoothera dauma (Scaly thrush). This new atadenovirus was discovered by metagenomic sequencing without virus cultivation. Analyses of the full genome sequence revealed that this new virus is a distinct member of the genus Atadenovirus and represents a novel species. ZdAdV has a genome of 34,760 bp with 28 predicted genes and 39% GC content. ZdAdV is the first atadenovirus to contain ORF19, a gene previously found only in aviadenoviruses. Phylogenetic analysis of ORF19 suggests that it was acquired by ZdAdV through horizontal gene transfer from an aviadenovirus. By analyzing all orthologous genes of aviadenovirus, mastadenovirus, atadenovirus, and siadenovirus, we also found potential horizontal gene transfer for the E4 gene in Pigeon aviadenovirus B. Our study widens our knowledge concerning the genetic diversity and evolutionary history of atadenoviruses and their potential for cross-species transmission.

Immunoprotection of FliBc chimeric fiber2 fusion proteins targeting dendritic cells against Fowl adenovirus serotype 4 infection.

Hepatitis-hydropericardium syndrome (HHS) is a highly fatal disease in chickens caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4), which has severe economic consequences. The fiber2 protein exhibits excellent potential as a candidate for a subunit vaccination against FAdV-4. Despite having a high safety profile, subunit vaccines have low immunogenicity due to their lack of infectivity, which leads to low levels of immune response. As a vaccine adjuvant, Salmonella flagellin possesses the potential to augment the immunological response to vaccinations. Additionally, a crucial strategy for enhancing vaccine efficacy is efficient presentation of immune antigens to dendritic cells (DC) for targeted vaccination. In this study, we designed FAdV-4-fiber2 protein, and a recombinant protein called FliBc-fiber2-SP which based on FAdV-4-fiber2 protein, was generated using the gene sequence FliBc, which retains only the conserved sequence at the amino and carboxyl termini of the flagellin B subunit, and a short peptide SPHLHTSSPWER (SP), which targets chicken bone marrow-derived DC. They were separately administered via intramuscular injection to 14-day-old specific pathogen-free (SPF) chickens, and their immunogenicity was compared. At 21 d postvaccination (dpv), it was found that the FliBc-fiber2-SP recombinant protein elicited significantly higher levels of IgG antibodies and conferred a vaccine protection rate of up to 100% compared to its counterpart fiber2 protein. These results suggest that the DC-targeted peptide fusion strategy for flagellin chimeric antigen construction can effectively enhance the immune protective efficacy of antigen proteins.

Epidemiological investigation of fowl adenovirus (FAdV) infections in ducks and geese in Shandong Province, China.

RESEARCH HIGHLIGHTS: Samples of suspected FAdV-infected waterfowl from farms in Shandong Province were collected from 2019 to 2022.Single infections with FAdV were less frequent than mixed infections.477 out of 792 samples (60.23%) tested positive for FAdV nucleic acids.Detection rate of FAdV was 65.47% in fattening duck farms, 55.73% in breeder duck farms and 54.55% in fattening geese farms.

Comparison of Penton and Hexon Genes of Fowl Adenovirus Serotype 11 in Molecular Detection of IBH in Broilers.

Fowl Adenoviruses (FAdVs) are widely distributed pathogens across the globe. The FAdVs from serotypes FAdV 2, 3, 8a, 8b, 9, and 11 are responsible for inclusion body hepatitis (IBH). Recently, increased mortality and IBH-suspected lesions were observed in 8-10-day-old broiler chickens in West Azerbaijan Province, Iran. In this regard, the present study aimed to compare penton and hexon genes of ADDV11 in the molecular detection of IBH in broiler chickens. In total, 100 liver specimens were collected from 10 suspected farms, and their DNAs were extracted. Two polymerase chain reactions (PCRs) were applied; one targeting the L1 region of the hexon gene and another aiming at the penton gene. Based on the findings, 60% of samples showed positive results in both PCRs and phylogenetic analysis clustered the studied viruses into serotype 11 (species D) FAdV. The detected FAdVs also shared a multitude of homologies with previously published serotype 11 viruses from Iran and those identified in Pakistan, Saudi Arabia, India, China, and Canada. This research not only provides an update on circulating FAdVs in Iran, but also introduces the penton gene as an alternative target for IBH diagnosis. Considering that IBH is a primary disease in Iran with both horizontal and vertical routes of transmission, urgent preventive measures are needed.

Efficient cross-protection against serotype 4/8a fowl adenoviruses (FAdVs): recombinant FAdV-4 with FAdV-8a Fiber.

IMPORTANCE: Epidemiological data reveal that FAdV-4 and FAdV-8a are the dominant serotypes of FAdVs in the poultry industry in China. Although three commercial inactivated vaccines against FAdV-4 have been licensed in China, the bivalent vaccine against both FAdV-4 and FAdV-8a is not available. Here, we used CRISPR-Cas9 and Cre-LoxP system to generate a recombinant virus FAdV4-F/8a-rF2 expressing the Fiber of FAdV-8a. Notably, FAdV4-F/8a-rF2 was highly attenuated and could provide efficient protection against both FAdV-4 and FAdV-8a in the chicken infection model, highlighting the applaudable application of FAdV4-F/8a-rF2 as a novel live-attenuated bivalent vaccine against the diseases caused by the infection of FAdV-4 and FAdV-8a.

Evidence of vertical transmission of fowl adenovirus 8b in ducks.

Fowl adenovirus mainly causes hydropericardium hepatitis syndrome (HHS), inclusion body hepatitis (IBH) and gizzard erosion (GE), etc. In 2015, the first outbreak of HHS was reported in broiler chickens in central China, followed by an outbreak in waterfowl. The first outbreak of HHS in broiler flocks in central China in 2015, followed by outbreaks in waterfowl, has severely restricted the healthy development of the poultry industry. During the investigation, fowl adenovirus was detected in ducklings from a total of seven hatcheries in Shandong, Inner Mongolia and Jiangsu provinces. In addition, the DNA of fowl adenovirus was detected in breeding ducks and their progeny. To test the hypothesis that FAdV can be transmitted vertically, sixty 250-day-old Cherry Valley breeder ducks were divided equally into three groups for experimental infection. FAdV-8b SDLY isolate (duck/Shandong/SDLY/2021, SDLY) preserved in our laboratory was injected intramuscularly into group A and inoculated orally into group B. FAdV-8b DNA was detected in the yolk membranes, embryos and allantoic fluid of duck embryos in the FAdV-infected group after inoculation. In addition, the FAdV-8b hexon gene isolated from yolk membranes, embryos, allantoic fluid and duck eggs was close to 100% nucleotide homology to the FAdV-8b hexon gene isolated from laying duck ovaries, indicating that fowl adenovirus can be transmitted vertically in ducks. These findings provide evidence for the possible vertical transmission of fowl adenovirus from breeder ducks to ducklings.

Visual detection of fowl adenovirus serotype 4 via a portable CRISPR/Cas13a-based lateral flow assay.

The widespread occurrence of fowl adenovirus serotype 4 (FAdV-4)-induced hepatitis-hydropericardium syndrome (HHS) has led to significant economic losses for the poultry industry. A sensitive, accurate, and practical FAdV-4 diagnostic approach is urgently required to limit the incidence of the disease. In the present study, a practical method for detecting FAdV-4 was developed using the CRISPR/Cas13a system and recombinase-aided amplification. The approach was based on 37°C isothermal detection with visible results being achieved. The detection limit of the target gene with this approach was only 101 copies/µl, making it very sensitive and specific. Clinical samples fared well when tested with the Cas13a detection method. For identifying FAdV-4, this novel detection approach was found to be sensitive, specific, and effective.RESEARCH HIGHLIGHTS First study using the CRISPR/Cas13a-based lateral flow detection assay for FAdV-4 detection.The results can be observed by the naked eye.The developed assay could provide an alternative tool for detection of FAdV-4 with minimal equipment.

The isolation and characterizations of a duck adenovirus 1 causing Egg Drop Syndrome in ducks, China.

A fowl adenovirus variant designated as DAdV-JSXZ strain was isolated from the tissue specimen of fallopian tubes of a duck case, which was submitted from a 276-day-old Cherry valley breeding duck flock experienced egg-dropping syndromes in March 2022. Full-genome sequence of the DAdV-I JSXZ strain by next-generation sequencing revealed that the complete genome length of DAdV-JSXZ strain was 33,213 nucleotides and shared a high degree of nucleotide identity (97.0-99.4 %) with other DAdV-I reference strains. In pathogenicity studies, this isolated duck JSXZ strain reproduced similar egg-dropping symptoms in healthy breeding ducks, pathologic lesions of follicular hemorrhage, and the laid eggs in low fertilization and hatchability rates. Our research findings demonstrated that DAdV-I JSXZ strain was one of the causative agents of duck egg dropping syndrome in egg-laying ducks and could cause acute respiratory symptoms in ducklings.

Adenovirus type D and type E infection in broiler chickens: the effect on CD4 and CD8 T cell response, cytokines expression and their immunopathology.

1. A total of 150-day-old chicks were divided into three groups of 50 birds (G1-G3); G1 and G2 were orally inoculated at 1-day old with 0.5 ml of 107 TCID50/ml FAdV-D serotype 2 (MT386509.1) and FAdV-E serotype 8a (MW847902), respectively, and G3 was blank control group.2. Cell-mediated immune response was evaluated by detection of CD4, CD8 T lymphocytes and the mRNA expression of IL6 and IL8 in the chicken spleen using q-PCR. Additionally, immunopathology was performed at 3, 5 and 7 day post infection (dpi) and weekly until the end of the experiment.3. Results revealed that transcription of inflammatory cytokines (IL6, IL8) was up regulated in the spleen of FAdV type D and type E infected chickens at various time points relative to the control group. A marked decrease in the number of CD4 and CD8 T lymphocytes at 5 and 7 dpi in G1 of chickens infected with FAdV type D. Whereas, in chickens infected with FAdV type E, the CD4 and CD8 T lymphocytes were markedly decreased at 7 dpi.4. In contrast, there were no significant differences in humoral immune responses against NDV vaccine in (G1 and G2) at different intervals post-vaccination compared to the control group. The histopathology of the bursa, thymus, and spleen in the infected groups showed lymphocytolysis with severe reticular cells hyperplasia and lymphoid depletion.5. In conclusion, fowl adenovirus types D and E have an immunosuppressive effect in broilers which may be considered one of the main causes of the continuous co-infections with other viruses reported in the field during the last 10 years.

Pathogenicity and innate immune responses induced by fowl adenovirus serotype 8b in specific pathogen-free chicken.

Fowl adenovirus serotype 8b (FAdV-8b), as causative agent of inclusion body hepatitis (IBH), poses a great threat to the poultry industry. Considering the importance of innate immune response in host against viral infections, we investigated pathogenicity of a FAdV-8b strain HLJ/151129 in 1-mo-old specific pathogen-free (SPF) chickens and immune responses of host to FAdV-8b infection in this study. The results demonstrated that no obvious clinical signs were observed in infected birds. Neither mobility nor mortality was observed in both FAdV-8b infected and control chickens, as well. However, hepatic necrosis and a small amount of inflammatory cell infiltration were observed by pathological analysis. Viral load was detected in bursa of Fabricius, cecal tonsils, liver, heart, spleen, Harderian glands, and thymus. Virus shedding and viremia generated as early as 3 days postinfection (dpi) (9/10) and reached the peak at 7 dpi (10/10). In addition, the infected birds had developed FAdV-specific antibodies at 7 dpi, and the antibody titers reached the peak at 14 dpi. Furthermore, the results demonstrated that the mRNA expression levels of most of toll-like receptors (TLRs), most of avian ß-defensins (AvBDs), and cytokines [interleukin (IL)-2, IL-6, and interferon (IFN)-γ], were significantly upregulated in most tissues at early phases of FAdV-8b infection, especially in liver and spleen. In contrast, FAdV-8b infection results in downregulation of TLR4, TLR5, and TLR21 expressions in some tissues of infected chickens. In addition, FAdV-8b infection upregulated myeloid differentiation factor 88 (MyD88), nuclear factor-kappa B (NF-κB) p65, and TIR-domain-containing adapter inducing interferon-ß (TRIF) expression in some tissues, while decreased NF-κBp65 and TRIF in spleen at both 72 hpi and 21 dpi. Taken together, these results confirmed that FAdV-8b could replicate in all investigated tissues of infected birds, and then, result in production of FAdV-specific antibody titers. Meanwhile, the FAdV-8b infection induces strong innate immune responses at early stage in chickens, which may associate with the viral pathogenesis.

A novel recombinant serotype 4 fowl adenovirus expressing fiber-2 protein of duck adenovirus 3.

Recently, the highly pathogenic serotype 4 fowl adenovirus (FAdV-4) and duck adenovirus 3 (DAdV-3) were outbroken and widespread, causing substantial economic losses to the duck industry. Therefore, there is an urgent need to generate a recombinant genetic engineering vaccine candidate against both FAdV-4 and DAdV-3. In this study, a novel recombinant FAdV-4 expressing the Fiber-2 protein of DAdV-3, designated as rFAdV-4-Fiber-2/DAdV-3, was generated based on CRISPR/Cas9 and Cre-LoxP systems. Indirect immunofluorescence assay (IFA) and western blot (WB) showed that the Fiber-2 protein of DAdV-3 in rFAdV-4-Fiber-2/DAdV-3 was expressed successfully. Moreover, the growth curve revealed that rFAdV-4-Fiber-2/DAdV-3 replicated efficiently in LMH cells and even showed a stronger replication ability compared to the wild type FAdV-4. The generation of the recombinant rFAdV-4-Fiber-2/DAdV-3 provides a potential vaccine candidate against both FAdV-4 and DAdV-3.

TaqMan probe-based qPCR method for specific detection and quantification of fowl adenovirus 8b challenge from chickens inoculated with live attenuated or inactivated virus.

Background: Fowl adenovirus (FAdV) 8b and other serotypes cause inclusion body hepatitis (IBH) in chickens. Specific detection of aetiologic serotype in mixed infection and vaccine failure could be difficult. Aim: The objective of this study was to develop a TaqMan probe-based qPCR method for the detection and quantification of the FAdV 8b challenge virus. Methods: Forty-eight broiler chickens inoculated with live attenuated or inactivated FAdV 8b strains at day 1 of age either with or without booster at day 14 post-inoculation were used. The chickens were challenged with a pathogenic strain of FAdV 8b at day 28 of age. Liver and cloacal swabs were collected on days 7 and 14 post-challenge. Primers and probes were designed, specificity confirmed, and used to carry out qPCR amplification. Results: The assay amplified the FAdV DNA challenge virus, but not that of the live attenuated virus. It could detect FAdV 8b DNA as low as 0.001 ng/µl in liver and cloacal swab samples. Copy numbers obtained indicate virus load and shedding. Conclusions: It shows that a selective detection of FAdV 8b within serotype is possible. It can be useful for rapid detection and diagnosis of the disease, virus quantification and differentiation within species, determination of vaccination failure, and efficacy especially the virus load in the target organ and shedding.

Transcriptome analysis reveals critical factors for survival after adenovirus serotype 4 infection.

Fowl adenovirus serotype-4 (FAdV-4) is highly lethal to poultry, making it one of the leading causes of economic losses in the poultry industry. However, a small proportion of poultry can survive after FAdV-4 infection. It is unclear whether there are genetic factors that protect chickens from FAdV-4 infection. Therefore, the livers from chickens uninfected with FAdV-4 (Normal), dead after FAdV-4 infection (Dead) or surviving after FAdV-4 infection (Survivor) were collected for RNA-seq, and 2,649 differentially expressed genes (DEGs) were identified. Among these, many immune-related cytokines and chemokines were significantly upregulated in the Dead group compared with the Survivor group, which might indicate that death is related to an excessive inflammatory immune response (cytokine storm). Subsequently, the KEGG results for DEGs specifically expressed in each comparison group indicated that cell cycle and apoptosis-related DEGs were upregulated and metabolism-related DEGs were downregulated in the Dead group, which also validated the reliability of the samples. Furthermore, GO and KEGG results showed DEGs expressed in all three groups were mainly associated with cell cycle. Among them, BRCA1, CDK1, ODC1, and MCM3 were screened as factors that might influence FAdV-4 infection. The qPCR results demonstrated that these 4 factors were not only upregulated in the Dead group but also significantly upregulated in the LMH cells after 24 h infection by FAdV-4. Moreover, interfering with BRCA1, CDK1, ODC1, and MCM3 significantly attenuated viral replication of FAdV-4. And interfering of BRCA1, CDK1, and MCM3 had more substantial hindering effects. These results provided novel insights into the molecular changes following FAdV-4 infection but also shed light on potential factors driving the survival of FAdV-4 infection in chickens.

Recombinant fiber-1 protein of fowl adenovirus serotype 4 induces high levels of neutralizing antibodies in immunized chickens.

Virulent fowl adenovirus serotype 4 (FAdV-4) causes hydropericardium syndrome (HPS) with high mortality in chickens, leading to significant economic losses to the poultry industry. The development of an effective vaccine is essential for successful disease control. Here, we produced recombinant fiber-1 protein of FAdV-4, isolated from a Japanese HPS outbreak strain, JP/LVP-1/96, using a baculovirus expression system and evaluated its immunogenicity and protective efficacy. Recombinant fiber-1 protein induced high levels of neutralizing antibodies in immunized chickens, which were maintained for a minimum of 10 weeks. After being challenged with the virulent FAdV-4 strain JP/LVP-1/96, the immunized chickens did not exhibit clinical signs of infection or histopathological changes, there was a significant reduction in the viral load in various organs and total serum proteins, and albumin levels did not decline. These results suggest that the recombinant fiber-1 protein produced in this study can serve as a subunit vaccine to control HPS in chickens.

Multiantigen epitope fusion recombinant proteins from capsids of serotype 4 fowl adenovirus induce chicken immunity against avian Angara disease.

Avian Angara disease caused by fowl adenovirus serotype 4 (FAdV-4) has spread widely and brought economic losses to the poultry industry in some countries. Effective vaccines for Angara disease control are currently lacking. In this study, four capsid proteins (hexon, penton, fiber1 and fiber2) from FAdV-4 were selected, and their optimal efficient antigenic epitopes predicted by bioinformatics software were tandemly linked with the flexible linker GGGGS. Based on their amino acid sequences, the DNA sequences for the genes encoding the multiantigen epitope tandem proteins (MAETPs) FAdV4:F1, FAdV4:P, FAdV4:F2 and FAdV4:H were chemosynthesized and then ligated by T4 ligases at the cleavage sites of restriction endonucleases to construct DNAs encoding the multilinked fusion recombinant proteins (MLFRPs) used as protective antigens from avian Angara disease. These genes ligated into the expression vector pET-28a were successfully expressed using the Escherichia coli prokaryotic expression system to prepare five kinds of MLFRPs (FAdV4:F1-P-F2-H, FAdV4:F1-F2-P-H, FAdV4:F1-F2-H-P, FAdV4:F1-P-H-F2 and FAdV4:F1-H-F2-P) for use to immunize chicks. FAdV-4 was injected into MLFRP-immunized chickens, and the challenge protection rate was evaluated. FAdV4:F1-P-F2-H produced the best protection against FAdV-4, with a single immunization resulting in a 100 % protection rate, followed by FAdV4:F1-F2-P-H (83.33 %) and FAdV4:F1-F2-H-P (66.67 %). FAdV4:F1-P-H-F2 and FAdV4:F1-H-F2-P were not able to induce a good immune protection effect after one immunization. However, all of the MLFRPs were capable of protecting the host from FAdV-4 infection after two immunizations. In conclusion, these MLFRPs generated based on capsid proteins of FAdV-4 are promising candidate subunit vaccines against Angara disease.

Otus scops adenovirus: the complete genome sequence of a novel aviadenovirus discovered in a wild owl.

We present the complete genome sequence of an aviadenovirus obtained by metagenomics from cloacal swabs taken from a free-living Eurasian scops owl (Otus scops, a small raptor distributed in Europe and several parts of Asia) in China. Thirty protein coding genes were predicted in this 40,239-bp-long genome, which encodes the largest fiber protein among all reported aviadenoviruses. The viral genome sequence is highly divergent, and the encoded proteins have an average of only 55% amino acid sequence identity to those of other adenoviruses. In phylogenetic analysis, the new owl virus grouped with members of the genus Aviadenovirus and formed a common clade with another owl adenovirus reported previously in Japan. This is the second complete genome sequence of an aviadenovirus discovered in owls, and its proteins have an average of 62% amino acid sequence identity to those of the previously reported owl adenovirus. Combining this result with comparative genomic analysis of all aviadenoviruses, we propose that this owl virus and the previously described Japanese owl adenovirus can be assigned to two new species in the genus Aviadenovirus. This study provides new data on the diversity of aviadenoviruses in wild birds.