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.

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.

Research Note: Overview of fowl adenovirus serotype 4: structure, pathogenicity, and progress in vaccine development.

Fowl adenovirus serotype 4 (FAdV) is highly pathogenic and lethal to chickens, especially broilers, which has emerged as one of the most important economic losses for the poultry industry in the past few years. Although inactivated vaccines have been widely used to control FAdV diseases, with the passage of time and the advancement of technology, live attenuated vaccines and subunit vaccines have also been developed, which are more attractive and effective vaccine candidates. This is an overview of avian adenoviruses, especially FAdV, which is related to the structure, pathogenicity of adenoviruses in birds, development and strategies used to make and use vaccines using different methods. As well as during this study it was determined that various vaccines against the new FAdV-4 genotype have been developed and many advances have been made in control disease However, many studies conducted in this field need extensive investigation.

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.

Variation in the affinity of three representative avian adenoviruses for the cellular coxsackievirus and adenovirus receptor.

According to previous studies, three representative avian adenoviral strains utilize coxsackievirus-adenovirus receptor (CAR) as a receptor and seem to exhibit diverse binding affinities and modes. Thus, further revealing the exact molecular mechanism underlying the interaction between different FAdVs and the attachment receptor CAR is necessary. In this study, we successfully solved the crystal structure of the FAdV-4 fiber1 knob at 1.6 Šresolution. The interaction between the fibre knob and different domains of CAR was verified by confocal microscopy, coimmunoprecipitation and surface plasmon resonance (SPR) analysis. The fibre knobs of the three representative fowl adenoviruses specifically recognized CAR domain 1 (D1), but the recognition of CAR domain 2 (D2) by chicken embryo lethal orphan (CELO) strains was weak. These results provide insights into the differences in adenovirus‒host cell interactions and have important implications for the exploration of viral invasion mechanisms.

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.

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.

Prevalence, genomic characteristics, and pathogenicity of fowl adenovirus 2 in Southern China.

In recent years, the occurrence of fowl adenovirus 2 (FAdV-2) has been on the rise in China, posing a significant threat to the poultry industry. This study aimed to investigate the epidemiology, phylogenetic relationship, genomic characteristics, and pathogenicity of FAdV-2. The epidemiological analysis revealed the detection of multiple FAdV serotypes, including FAdV-1, FAdV-2, FAdV-3, FAdV-4, FAdV-8a, FAdV-8b, and FAdV-11 serotypes. Among them, FAdV-2 exhibited the highest proportion, accounting for 21.05% (8/38). The complete genomes of these 8 FAdV-2 strains were sequenced. Genetic evolution analysis indicated that these FAdV-2 strains formed a separate branch within the FAdV-D group, sharing 94.60 to 97.90% nucleotide similarity with the reference FAdV-2 and FAdV-11 strains. Notably, the recombination analysis revealed that 5 out of the 8 FAdV-2 strains, exhibited recombination events between FAdV-2 and FAdV-11. The recombination regions involved Hexon, Fiber, ORF19 genes and 3' end. Furthermore, pathogenicity experiments demonstrated that recombinant FAdV-2 XX strain is capable of inducing mortality rate of 66.70% and causing more severe hepatitis hydropericardium syndrome (HHS) in 6-wk-old specific-pathogen-free chickens. These findings contribute to our understanding of the prevalence, genomic characteristics, and the pathogenicity of FAdV-2, providing foundations for FAdV-2 vaccine development.

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.

Study on Propagation and Adaptation of EDS-76 Avian Adenovirus in Duck and SPF Primary Embryonic Chicken Cell Culture Comparison to Duck and SPF Embryonated Chicken Eggs.

Egg drop syndrome (EDS) is a major viral infectious poultry disease with severe economic losses in laying hens. The disease is caused by an adenovirus and can be transmitted horizontally and vertically. This study investigated the EDS virus (EDSV) infection in duck embryo fibroblasts (DEF), specific pathogen-free (SPF) embryo fibroblasts, and SPF egg embryos using different methods. The results were compared to the virus culture in duck and SPF chicken eggs. Duck and chicken fibroblast cells were used as the primary cell culture in Dulbecco's Modified Eagle Medium, and the low-pathogenic duck adenovirus was used to infect the ducks and SPF fibroblasts primary cell cultures, as well as the duck and SPF eggs. The titer of the virus was measured by hemagglutination assay, ECID50, plaque-forming unit, and TCID50 methods. The results revealed that EDSV could proliferate in the chorioallantoic membrane of DEF cells and duck eggs, compared to the chorioallantoic membrane of chicken embryo fibroblasts (CEF) and SPF chicken eggs. The findings showed that duck egg embryos and primary DEF cell lines are more appropriate for EDSV replication, compared to CEF and SPF chicken eggs. This suggests that the use of DEF culture for producing avian adenovirus EDS-76 is a suitable alternative for the embryonic egg culture.

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.

FAdV-4 Promotes Expression of Multiple Cytokines and Inhibits the Proliferation of aHEV in LMH Cells.

Single or mixed infections of multiple pathogens such as avian hepatitis E virus (aHEV) and avian leukosis virus subgroup J (ALV-J) have been detected in numerous laying hens with severe liver injury in China. Thus, aHEV and immunosuppressive viruses are speculated to cause co-infections. In this study, co-infection with aHEV and fowl adenovirus (FAdV) was confirmed by nested RT-PCR and recombinase-aided amplification combined with gene sequencing in two flocks with severe liver injury. Subsequently, the two reference strains, aHEV and FAdV-4, were inoculated into LMH cells to identify their co-infection potential. Confocal microscopy revealed aHEV and FAdV-4 co-infected LMH cells. In addition, the replication dynamics of aHEV and FAdV-4 along with the expression levels of immuno-cytokines were measured. The results indicated colocalization of aHEV and FAdV-4 and inhibition of viral replication in LMH cells. The transcription levels of MDA5, Mx, OASL, and IFN-α were significantly upregulated in LMH cells, whereas those of immune-related factors induced by FAdV-4 were downregulated upon FAdV-4 and aHEV co-infection. These results confirmed the co-infection of aHEV and FAdV-4 in vitro and prompted the antagonistic pathogenic effects of FAdV-4 and aHEV, thereby providing novel insights into the counterbalancing effects of these viruses.

Characterisation of a novel aviadenovirus associated with disease in tawny frogmouths (Podargus strigoides).

Aviadenoviruses are widespread in wild birds but rarely cause disease in nature. However, when naïve species are exposed to poultry or aviaries, aviadenoviruses can lead to disease outbreaks. This study characterised a novel aviadenovirus infection in a native Australian bird, the tawny frogmouth (Podargus strigoides) during an outbreak investigation. The identified complete genome of aviadenovirus, named tawny frogmouth aviadenovirus A (TwAviAdV-A) was 41,175 bp in length containing 52 putative genes. TwAviAdV-A exhibits the common aviadenovirus genomic organisation but with a notable monophyletic subclade in the phylogeny. The TwAviAdV-A virus was hepatotrophic and the six frogmouths presented to the wildlife hospitals in South Eastern Queensland most commonly exhibited regurgitation (in four frogmouths). Three were died or euthanized, two recovered, and one showed no signs. The detection of TwAviAdV-A in frogmouths coming into care re-emphasizes the need for strict biosecurity protocols in wildlife hospitals and care facilities.

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.

Genomic and Pathologic Characterization of the First FAdV-C Serotype 4 Isolate from Black-Necked Crane.

Fowl adenoviruses (FAdVs) are distributed worldwide in poultry and incriminated as the etiological agents for several health problems in fowls, and are capable of crossing species barriers between domestic and wild fowls. An FAdV strain was, for the first time, isolated from black-necked crane in this study, and was designated as serotype 4 Fowl aviadenovirus C (abbreviated as BNC2021) according to the phylogenetic analysis of its DNA polymerase and hexon gene. The viral genomic sequence analysis demonstrated that the isolate possessed the ORF deletions that are present in FAdV4 strains circulating in poultry fowls in China and the amino acid mutations associated with viral pathogenicity in the hexon and fiber 2 proteins. A viral challenge experiment with mallard ducks demonstrated systemic viral infection and horizontal transmission. BNC2021 induced the typical clinical signs of hepatitis-hydropericardium syndrome (HHS) with swelling and inflammation in multiple organs and showed significant viral replication in all eight organs tested in the virus-inoculated ducks and their contactees at 6 dpi. The findings highlight the importance of surveillance of FAdVs in wild birds.

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.

Recombinant Fowl aviadenovirus E (FAdV-E) penton base vaccination fails to confer protection against inclusion body hepatitis (IBH) in chickens.

Inclusion body hepatitis (IBH) is a metabolic disease affecting chickens, associated with different serotypes of fowl adenovirus (FAdV). Experimentally tested vaccines against IBH include several capsid-based subunit vaccines, but not the penton base protein. In the present study, specific pathogen-free chickens were vaccinated with recombinant penton base expressed from each of two different FAdV serotypes (FAdV-7 and FAdV-8b), followed by challenge with a virulent IBH-causing strain. No protection was observed with either vaccine, possibly due to the low immunogenicity of each protein and their inability to induce neutralizing antibodies in the host.

Virome Profiling of Chickens with Hepatomegaly Rupture Syndrome Reveals Coinfection of Multiple Viruses.

Liver diseases seriously challenge the health of chickens raised on scaled farms and cause tremendous economic losses to farm owners. The causative agents for liver diseases are still elusive, even though various pathogens, such as the hepatitis E virus, have been reported. In the winter of 2021, a liver disease was observed on a chicken farm in Dalian, China, which increased chicken mortality by up to 18%. We conducted panvirome profiling of the livers, spleens, kidneys, and recta of 20 diseased chickens. The viromic results revealed coinfection of multiple viruses, including pathogenic ones, in these organs. The viruses were highly identical to those detected in other provinces, and the vaccine and field strains of avian encephalomyelitis virus (AEV) and chicken infectious anemia virus (CIAV) cocirculated on the farm. In particular, the liver showed higher abundance of AEV and multiple fowl adenoviruses than other organs. Furthermore, the liver also contracted avian leukemia virus and CIAV. Experimental animals with infected liver samples developed minor to medium lesions of the liver and showed a virus abundance profile for AEV across internal organs similar to that in the original samples. These results suggest that coinfection with multiple pathogenic viruses influences the occurrence and development of infectious liver disease. The results also highlight that strong farm management standards with strict biosafety measures are needed to minimize the risk of pathogenic virus introduction to the farm.