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.

Survey of selected viral agents (herpesvirus, adenovirus and hepatitis E virus) in liver and lung samples of cetaceans, Brazil.

Hepatic and pulmonary lesions are common in cetaceans, despite their poorly understood viral etiology. Herpesviruses (HV), adenoviruses (AdV) and hepatitis E virus (HEV) are emerging agents in cetaceans, associated with liver and/or pulmonary damage in mammals. We isolated and molecularly tested DNA for HV and AdV (n = 218 individuals; 187 liver and 108 lung samples) and RNA for HEV (n = 147 animals; 147 liver samples) from six cetacean families. All animals stranded or were bycaught in Brazil between 2001 and 2021. Positive-animals were analyzed by histopathology. Statistical analyses assessed if the prevalence of viral infection could be associated with the variables: species, family, habitat, region, sex, and age group. All samples were negative for AdV and HEV. Overall, 8.7% (19/218) of the cetaceans were HV-positive (4.8% [9/187] liver and 11.1% [12/108] lung), without HV-associated lesions. HV-prevalence was statistically significant higher in Pontoporiidae (19.2%, 10/52) when compared to Delphinidae (4.1%, 5/121), and in southeastern (17.1%, 13/76)-the most industrialized Brazilian region-when compared to the northeastern region (2.4%, 3/126). This study broadens the herpesvirus host range in cetaceans, including its description in pygmy sperm whales (Kogia breviceps) and humpback whales (Megaptera novaeangliae). Further studies must elucidate herpesvirus drivers in cetaceans.

Isolation, characterization, evaluation of pathogenicity, and immunomodulation through interferon production of duck adenovirus type-3 (DAdV-3).

Duck adenovirus type-3 (DAdV-3) is a poorly characterized duck virus. A comprehensive analysis of the DAdV-3 pathogenicity and host immune response could be a valuable addition. Herein, DAdV-3 was isolated from Muscovy duck and virus-specific genes were confirmed by polymerase chain reaction (PCR). The obtained gene fragments were sequenced and compared with the reference sequence. Results confirmed that the clinically isolated virus was DAdV-3, named as HF-AN-2020. To evaluate DAdV-3 host immune response, the expression levels of MDA5, STING, IRF7, MAVS, and NF-κB, and inflammatory cytokines (IFN-ß, IFN-γ, and IL-1ß) were determined by quantitative reverse transcriptase PCR (qRT-PCR). The expression levels of IFN-ß and IFN-γ were 32.6- and 28.6-fold, respectively, higher (P < 0.01) than the control group. It was found that the upregulation of STING and NF-κB pathways was directly involved in the regulation of inflammatory cytokines (IFN-ß, IFN-γ, and IL-1ß). Furthermore, the gene regulation pathways consecutively upregulated the expression levels of MDA5, STING, IRF7, MAVS, and NF-κB up to 31.6, 10.5, 31.4, 2.2, and 2.6-fold, respectively, higher (P < 0.01) than the control group. The TCID50 of DAdV-3 for Muscovy duck and chicken was 10-3.24/0.1 mL with 0% mortality, indicating low pathogenicity in both Muscovy ducks and chickens, but DAdV-3 can induce higher expression of interferons. Genome analysis showed mutations in 4 amino acids located in ORF19B (Ser to Thr), ORF66 (Leu to Phe, Ile to Leu), and ORF67 (Gly to stop codon). This study provides essential and basic information for further research on the mechanism of the cellular immune responses against adenoviruses.

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.

Identification of a novel adenovirus in liver tissue sample of the Great Himalayan leaf-nosed bat (Hipposideros armiger).

Bats are important reservoirs for many zoonotic viruses. To explore and monitor potential novel viruses carried by bats, 21 liver samples of bats (Hipposideros armiger) were collected from Yunnan Province in southern China. Only one (4.8%) of all models was detected with adenovirus. The whole genome strain obtained by the viral metagenomics method combined with PCR was temporarily named YN01. The complete genome of YN01 was 37,676 bp, with a G + C content of 55.20% and 28 open reading frames. Phylogenetic analysis indicated that the strain YN01 can be classified as genus Mastadenovirus and was the most similar to the adenovirus isolated from Rhinolophus sinicus in China in 2016. The analysis is needed to verify the possibility of cross-species transmission. This virological investigation has increased our understanding of the ecology of bat-borne viruses in this area and provided a reference for possible future infectious diseases.

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.

Identification of ORF1B as a unique nonstructural protein for fowl adenovirus serotype 4.

Fowl adenovirus serotype 4 (FAdV-4), the causative agent of hepatitis-hydropericardium syndrome (HHS), is a double-stranded DNA virus. Although many structural proteins have been deeply studied, the coding potential of some other open reading frames (ORFs) and the biological functions of their products during virus infection have not been fully elucidated. Here, a unique nonstructural protein ORF1B of FAdV-4 was identified and its expression kinetics along infection was analyzed. Except that of FAdV-10, a member of the same genus as FAdV-4, FAdV-4 ORF1B shared as low homologous identity as 29.2% in amino acid sequence with the other ten counterparts. Structurally, ORF1B was mapped on the N-terminal region of the genome between 1485 nt to 1808 nt and predicted to only contain two α-helix. Confocal immunofluorescence assay with homemade rabbit polyclonal antibody demonstrated that ORF1B could be simultaneously observed with structural protein Fiber 1 in FAdV-4-infected cells. Western blot further showed that ORF1B could only be detected in the infected cells but not mature virions, suggesting ORF1B was a nonstructural protein. Subsequently, the expression level of ORF1B detected by qRT-PCR and IFA was gradually decreased along with FAdV-4 infection, suggesting ORF1B was an early gene transcript. These results will lay a solid foundation to further study the biological effect of ORF1B on the replication and pathogenicity of FAdV-4.

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.

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.

Naturally occurring psittacid alphaherpesvirus 3 and probable adenovirus coinfection in an Indian ringneck parakeet in the United States.

Psittacid alphaherpesvirus 3 (PsAHV-3) is a rarely reported virus that has been associated with pneumonia in psittacine birds. A 5-mo-old Indian ringneck parakeet (syn. rose-ringed parakeet; Psittacula krameri) was euthanized after developing torticollis and lethargy. Postmortem examination of the parakeet revealed severe pneumonia with syncytial cells containing eosinophilic intranuclear inclusion bodies (INIBs) in the respiratory epithelium. Gene sequencing of total DNA extraction detected PsAHV-3 in the frozen lung. The pancreas had severe lytic necrosis, with the remaining parenchymal cells expanded by large basophilic INIBs. Electron microscopy of the pancreas revealed intranuclear paracrystalline arrays of viral particles morphologically resembling adenovirus. Although we found 3 PsAHV-3 cases in birds in a literature search, we found no cases with adenoviral coinfection.

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.

Detection and Molecular Characterization of Animal Adenovirus and Astrovirus from Western Maharashtra, India.

Astroviruses (AstV) and adenoviruses (AdV) are associated with diarrhoea in young animals. However, the epidemiology and genetic diversity of AstVs and AdVs in animals is not well studied. Hence, the present study was conducted to detect and characterize AstVs and AdVs in calves, piglets and puppies from Western Maharashtra, India. Out of the processed porcine (48), canine (80), and bovine (65) faecal samples, the porcine AstV (PAstV), bovine AstV (BAstV), canine AstV (CAstV), and porcine AdV (PAdV) were detected in 12.5%, 7.69%, 3.75% and 4.1% of samples, respectively. In the RNA-dependent RNA polymerase region-based phylogenetic analysis, the detected BAstV strains grouped with MAstV-28, MAstV-33, and MAstV-35, CAstV strains belonged to MAstV-5; PAstV strains belonged to MAstV-24, MAstV-26, and MAstV-31. However, in hexon gene-based phylogeny, both the detected PAdV were of genotype 3, exhibiting 91.9-92.5% nucleotide identity with Ivoirian and Chinese strains. The study reports first-time BAstVs from calves and PAdV-3 from piglets in India. The study revealed diversity in the circulation of AstVs in tested animals and AdVs in pigs, and suggested that they alone might be associated with other diarrhoea or in combination with other enteric pathogens, thus highlighting the necessity of extensive epidemiological investigations to develop diagnostic tools and control measures.