Fowl Adenovirus Research Articles

CELO Fiber1 Knob Is a Promising Candidate to Modify the Tropism of Adenoviral Vectors.

Fowl adenovirus 4 (FAdV-4) has the potential to be constructed as a gene transfer vector for human gene therapy or vaccine development to avoid the pre-existing immunity to human adenoviruses. To enhance the transduction of FAdV-4 to human cells, CELO fiber1 knob (CF1K) was chosen to replace the fiber2 knob in FAdV-4 to generate recombinant virus F2CF1K-CG. The original FAdV4-CG virus transduced 4% human 293 or 1% HEp-2 cells at the multiplicity of infection of 1000 viral particles per cell. In contrast, F2CF1K-CG could transduce 98% 293 or 60% HEp-2 cells under the same conditions. Prokaryotically expressed CF1K protein blocked 50% transduction of F2CF1K-CG to 293 cells at a concentration of 1.3 µg/mL while it only slightly inhibited the infection of human adenovirus 5 (HAdV-5), suggesting CF1K could bind to human cells in a manner different from HAdV-5 fiber. The incorporation of CF1K had no negative effect on the growth of FAdV-4 in the packaging cells. In addition, CF1K-pseudotyped HAdV-41 could transduce HEp-2 and A549 cells more efficiently. These data indicated that CF1K had the priority to be considered when there is a need to modify adenovirus tropism.

β-catenin facilitates fowl adenovirus serotype 4 replication through enhancing virus-induced autophagy

β-catenin is a key component of the Wnt/β-catenin signal transduction cascade which is a highly conserved signaling pathway in eukaryotes. Increasing evidence suggests that the Wnt/β-catenin signaling pathway is involved in the infection of many viruses. However, its role in fowl adenovirus serotype 4 (FAdV-4) replication remains unclear. In the present study, we showed that FAdV-4 infection increased the expression of β-catenin and promoted the nuclear translocation of β-catenin. Overexpression of β-catenin and LiCl treatment stimulated the accumulation of β-catenin in the nucleus, and then facilitated FAdV-4 replication. Conversely, repression of β-catenin by inhibitors and siRNA significantly inhibited FAdV-4 replication. Furthermore, inhibition of autophagy by 3-Methyladenine (3-MA) suppressed the FAdV-4 replication, and repression of β-catenin inhibited the FAdV-4-triggered autophagy. In conclusion, the nuclear translocation of β-catenin benefits FAdV-4 replication, and suppression of β-catenin limits FAdV-4 production by inhibiting FAdV-4-induced autophagy. These findings indicated that β-catenin is an important regulator of FAdV-4 replication which can serve as a potential target for anti-FAdV-4 agents.

Pathomorphological changes in the liver of broiler chickens caused by adeno-virus infection. Description of a spontaneous case in a private farm of the Lviv region

The article covers the results of pathomorphological studies of broiler chickens' liver (Cobb 500 cross) spontaneously damaged by Group I fowl adenovirus. Clinical signs of the disease in broiler chickens appeared on the 15th day of life and were manifested by a sharp decrease in water and feed consumption. The fowl was lethargic for several days, breathing hard, mostly sitting with its head down and eyes closed. During the breeding period, the mortality rate reached 13.2 %, accompanied by significant growth retardation. A peak of mortality was observed within 20–24 days of life. Antibody titers monitored by immunoenzymatic method using BioChek ELISA tests for infectious bursal disease (IBD), infectious bronchitis (IBV), and Newcastle disease (ND) virus were within normal post-vaccination levels. Pathological autopsy of broiler chickens revealed hepatomegaly, diffuse punctate and petechial hemorrhages under the Glisson's capsule, and military necrosis in the liver parenchyma. Hydropericarditis, pulmonary edema, renal dystrophy, and catarrhal enteritis were also detected in some cases. Liver fragments were selected for histological examination, fixed in a 10 % aqueous solution of neutral formalin, washed in water, and dehydrated in an ascending series of alcohols, followed by sealing and embedding in paraffin. Deparaffinized sections were stained with Mayer's hematoxylin and eosin. Optical examination of hepatocytes revealed large, round basophilic intranuclear inclusion bodies, multifocal necrosis of hepatocytes, stasis, and fatty dystrophy. According to the molecular genetic test results of broiler chickens' liver fragments, the DNA of Group I fowl adenovirus, serotype FAdV-11, was isolated.

Efficacy, humoral, and cell-mediated immune response of inactivated fowl adenovirus 8b propagated in chicken embryo liver cells using bioreactor in broiler chickens.

Fowl adenovirus (FAdV) 8b causes inclusion body hepatitis, resulting in major economic losses globally among chickens. The objectives were to inactivate FAdV 8b isolate propagated in chicken embryo liver (CEL) cells using a stirred tank bioreactor (UPM08136P5B1) and determine the humoral and cell-mediated immune response, efficacy, and virus shedding in broiler chickens. The FAdV 8b isolate UPM08136P5B1 was inactivated using binary ethyleneimine, adjuvanted with Montanide 71VG, inoculated into day-old broiler chickens in a booster group (BG) and non-booster group (NBG), and challenged with a pathogenic FAdV 8b strain. Clinical signs, gross lesions, body weight (BW), liver: body weight ratio, FAdV antibody titer using enzyme-linked immunosorbent assay, and histopathological changes were recorded. The CD3+, CD4+, and CD8+ T-lymphocyte profiles of the liver, spleen, and thymus using flow cytometry, and viral load in liver and cloacal shedding using quantitative polymerase chain reaction were evaluated. Chickens in the challenged control group (CCG) exhibited mild clinical signs, gross lesions, and histopathological changes, which were absent in the inoculated groups, and had lower BW and higher liver BW ratio than chickens in the unchallenged control group (UCG); BG and NBG on 35- and 42-days post-inoculation (DPI). Chickens in NBG and BG had higher antibodies than UCG on 7, 21, 35, and 42 DPI. The challenged BG and NBG produced higher antibodies than the CCG on 35 DPI. T-lymphocytes were higher among the inoculated groups than UCG in the liver, spleen, and thymus. Inoculated challenged groups recorded higher CD3+, CD4+, and CD8+ T-lymphocytes on 35 and 42 DPI than CCG. The challenged control group had a significantly higher viral load in the liver than challenged that in BG on 35 DPI and BG and NBG on 42 DPI. The challenged control group had significantly higher challenge FAdV shedding than challenged inoculated groups on 35 and NBG on 42 DPI. UPM08136P5B1 was successfully inactivated and mixed with Montanide 71VG. The inactivated vaccine candidate that induced humoral and cellular immunity was effective, reduced FAdV load in the liver, and shedding in the cloaca, and could be useful against FAdV 8b infections in chickens.

Molecular Epidemiology of Fowl Adenovirus (FAdV) from Inclusion Body Hepatitis (IBH) Incidences from Indian Broilers Revealed the Prevalence of Serotypes of FAdV-D and E

Background: Inclusion body hepatitis (IBH) caused by Fowl adeno virus (FAdV), is a re-emerging threat to broilers causing considerable economic loss to poultry industry worldwide. Methods: The present study investigated the IBH outbreaks in commercial broiler flocks from different regions of India between 2019- and 2021 and identified the serotypes associated with it by molecular characterization of hexon gene of FAdV strains. Result: 42 out of 98 flocks affected with IBH tested FAdV positive by PCR. Phylogenetic analysis grouped these strains into three serotypes; 25 strains (67.56%) were clustered with FAdV-11 serotype, 11 strains (29.72%) with FAdV-8b serotype and one strain with FAdV-8a (2.70%). Three serotypes FAdV-11, 8a and 8b of species D and E are currently circulating in Indian broilers. The present study indicates the need for continuous surveillance of FAdV and provides useful information for devising vaccination strategies for effective control of IBH in chickens.

Phylogenetic and pathogenic characterization of current fowl adenoviruses in China.

In recent years, fowl adenoviruses (FAdVs) continue to outbreak and cause huge economic losses to the poultry industry in China. The homologous recombination accounts for the diversity serotypes of adenovirus. However, the prevalence, recombination and pathogenicity of current FAdVs remain unclear. Herein, the prevalence, phylogenetic feature and pathogenicity of FAdVs in China in 2019 were characterized. Our findings showed that multiple species and serotypes of FAdVs currently circulate in China, including A, C, D and E species, and 1, 2, 4, 8a and 8b serotypes. Notably, the recombination occurred between FAdV-8a and FAdV-8b, and the recombination regions included Hexon, Fiber, ORF19 and ORF20. All five FAdVs replicated effectively in various chicken tissues, and viral shedding peaked at 4-8 dpi. Except CH/GDSZ/1905(FAdV-1/A), the remaining FAdVs caused obvious inclusion body hepatitis (IBH) in 3-week-old specific-pathogen-free (SPF) chickens, of which CH/JSXZ/1905(FAdV-4/C) caused hydropericardium-hepatitis syndrome (HHS) with a mortality rate of 62.5%. Taken together, our findings illustrate the prevalence, recombination and pathogenicity of current FAdVs in China and strengthen surveillance and further pathogenicity studies of FAdVs are extremely urgent.

Molecular phylodynamics of fowl adenovirus serotype 11 and 8b from inclusion body hepatitis outbreaks.

Fowl adenovirus (FAdV) serotypes are involved in a variety of clinical manifestations in poultry and has resulted in substantial economic loss to the poultry farmers. Despite the endemicity of Inclusion body hepatitis (IBH) in South Asian countries, including India, its etiology is not well studied. In western India, the rural poultry flocks obtained from the vaccinated parents were experiencing disease outbreaks with substantial economic losses due to heavy outbreaks and mortality. Therefore, the study was conducted to decipher the molecular epidemiology of the FAdV from field outbreaks in western India. A total of 37 commercial broiler poultry flocks and 29 village poultry flocks of western India were visited during 2019 to 2021. Out of these, 19.14% flocks showed incidence of IBH during the age of 15 to 35days. The mortality ranged from 3.3 percent to 55.28 percent. The samples were subjected for amplification of partial hexon gene covering loop 1 and loop 2. The results revealed 48.28% positivity by PCR. The sequence analysis identified 14 isolates as species D serotype 11 with 0.97 to 0.99% divergence and two as species E serotype 8b with 0.99% divergence. The FAdV-11 isolates showed amino acid substitutions D195N, T399A, N417S, and N496H. The amino acids I188 and N195 were conserved in FAdV-11. The molecular clock in Bayesian methods was used to determine most common ancestor. The isolates MH379249 and MH379248 were determined the most recent common ancestor for FAdV-11 and FAdV-8b isolates. The analysis suggested evolution of 10 FAdV-11 strains in 2012, and four FAdV-11 strains and two FAdV-8b strains in 2018.

Illumina high-throughput sequencing for the genome of emerging fowl adenovirus D species and C species simultaneously

In recent years, clinical cases of inclusion body hepatitis (IBH) and hydropericardium syndrome (HPS) have been emerging and increasing in chicken flocks worldwide. Mixed infections with 2 or more fowl adenovirus (FAdV) serotypes were common in these cases. Herein, we collected a clinical sample that was positive for FAdV from 40-day-old broilers with IBH and HPS symptoms in Shandong province of China and determined the complete genome of FAdVs on the Illumina HiSeq4000 platform. The results showed that the sample contained 2 FAdV strains of D species and C species and named SD1763-1 and SD1763-2 respectively. The genome of SD1763-1 strain was 43,913 nt in length, with a G+C content of 53.51%, whereas SD1763-2 strain was 43,721 nt in length, with a G+C content of 54.87%. Sequence alignment and phylogenetic analysis revealed that strain SD1763-1 was clustered together with serotype 2/11 of FAdV-D, and SD1763-2 was clustered together with FAdV-4. There is no recombination between the genomes of the 2 viruses of FAdV-D and FAdV-C in the present study. This is the first report of obtaining 2 genomic sequences of FAdV strains simultaneously by direct use of deep sequencing in one clinical individual chicken sample, which provided direct evidence for mixed infections of adenovirus serotypes in the clinic and enriched the genome data to explore the geographic biomarkers and virulence signatures of the genus Aviadenovirus.

Local cellular immune response plays a key role in protecting chickens against hepatitis-hydropericardium syndrome (HHS) by vaccination with a recombinant fowl adenovirus (FAdV) chimeric fiber protein.

Fowl adenovirus (FAdV)-induced diseases hepatitis-hydropericardium syndrome (HHS) and inclusion body hepatitis (IBH) have been affecting the poultry industry with increasing severity in the last two decades. Recently, a subunit vaccine based on a chimeric fiber protein with epitopes from different fowl adenovirus serotypes (named crecFib-4/11) has been shown to confer simultaneous protection against both HHS and IBH. However, the underlying immune mechanisms in chickens are still enigmatic, especially because of frequently absent neutralizing response despite high levels of protection. In this study, we investigated the kinetics of the humoral and cellular immune responses in specific pathogen-free chickens after vaccination with crecFib-4/11 and/or challenge with a HHS-causing strain, on a systemic level, as well as locally in target and lymphoid organs. The humoral response was assessed via enzyme-linked immunosorbent assay (ELISA) and virus neutralization test in serum, while the cellular immune response was determined by phenotyping using flow cytometry. Although vaccination induced serum antibodies, as confirmed by ELISA, such antibodies exhibited no pre-challenge neutralizing activity against FAdV-4. Nevertheless, immunized birds experienced a significant B cell increase in the liver upon challenge, remaining high throughout the experiment. Furthermore, vaccination stimulated the proliferation of cytotoxic T lymphocytes, with earlier circulation in the blood compared to the challenge control and subsequent increase in liver and spleen. Overall, these findings imply that protection of chickens from HHS after crecFib-4/11 vaccination relies on a prominent local immune response in the target organs, instead of circulating neutralizing antibodies.

Detection and Molecular Characterization of Enteric Viruses in Poultry Flocks in Hebei Province, China.

Simple SummaryEnteric viruses act as etiological agents for a series of health disturbances that pose a threat to commercial chickens worldwide. The affected chickens exhibit stunted growth, low feed conversion, etc. On a global scale, research on enteric virus diversity has been performed in countries such as India, South Korea and Brazil, yet at present, there have been no conclusive reports of avian enteric viruses in China. In the present study, the virus species, infection types, clinical symptoms, and relationships among the virus species were studied in 145 positive enteric virus samples. Additionally, the evolutionary relationship and recombination of the viruses were also further studied. The results of this study can be used to define the distribution and infection type of enteric viruses in poultry, and to analyze the classification of and evolutionary relationship between certain viruses.Enteric viruses, as a potential pathogen, have been found to be vital causes of economic losses in poultry industry worldwide. The enteric viruses widely studied to date mainly include avian nephritis virus (ANV), avian reovirus (ARe), chicken astrovirus (CAstV), chicken parvovirus (ChPV), fowl adenovirus group I (FAdV-1), infectious bronchitis virus (IBV), and avian rotavirus (ARoV). This paper aimed to identify single and multiple infections of the seven enteric viruses using the data obtained from positive 145 enteric virus samples in poultry flocks from different areas in Hebei Province, throughout the period from 2019 to 2021. Next, the correlation between bird age and clinical signs was investigated using PCR and RT-PCR techniques. Furthermore, the whole genomes of seven parvovirus strains and open reading frame 2 (ORF2) of six CAstV strains and eight ANV strains were sequenced for phylogenetic analysis and recombination analysis, to characterize the viruses and evaluate species correlation and geographic patterns. A total of 11 profiles of virus combinations were detected; 191 viruses were detected in 145 samples; 106 single infections were reported in 73.1% of the samples; and multiple infections were detected in the remaining 26.9%. For viruses, 69% of ChPV was correlated with single infection, while ANV (61.4%) and CAstV (56.1%) were correlated with multiple infections. However, IBV and ARe were not detected in any of the samples. Recombination events were reported in parvovirus, and all CAstV sequences investigated in this paper were included within genotype Bii. The eight ANV strains pertained to different subtypes with significant differences. The above results revealed for the first time the complexity of enteric viruses over the past several years, thus contributing to disease prevention and control in the future.

Highly soluble and stable ‘insertion domain’ of the capsid penton base protein provides complete protection against infections caused by fowl adenoviruses

In the current study, we have evaluated the protective efficacy of the ‘insertion domain’ which is commonly found in the capsid penton base protein of many adenoviruses. Using the ‘insertion domain’ of the penton base protein of a representative fowl adenovirus, fowl adenovirus serotype 4 (FAdV-4), we find that the ‘insertion domain’ can readily be expressed in a soluble form in the bacterial system, and can be purified in sufficient quantities through simple chromatographic methods. We demonstrate that the ‘insertion domain’, when employed as a subunit vaccine candidate, provides complete protection against hydropericardium syndrome, caused by FAdV-4, in chickens. The data presented here indicate that the protein, adjuvanted with Montanide™ ISA71 VG, provides complete protection in chickens against a lethal FAdV-4 challenge after administration of two doses (100 μg of the protein per dose) two weeks apart (the first dose at the 7th day of life and a booster dose at the age of 21 days). Furthermore, the purified protein can be stored at low temperatures without any observable loss in the protein integrity up to one year, tested so far. Due to the conserved nature of the ‘insertion domain’ across the penton base protein of fowl adenoviruses, it is suggested that homologous insertion domains could be employed as highly stable and cost-effective subunit vaccine candidates against infections caused by respective fowl adenoviruses.

Epidemiology, pathology, prevention, and control strategies of inclusion body hepatitis and hepatitis-hydropericardium syndrome in poultry: A comprehensive review.

Infection with fowl adenoviruses (FAdVs) can result in a number of syndromes in the production of chicken, including inclusion body hepatitis (IBH), hepatitis-hydropericardium syndrome (HHS), and others, causing enormous economic losses around the globe. FAdVs are divided into 12 serotypes and five species (A–E; 1–8a and 8b−11). Most avian species are prone to infection due to the widespread distribution of FAdV strains. The genus aviadenovirus, which is a member of the adenoviridae family, is responsible for both IBH and HHS. The most popular types of transmission are mechanical, vertical, and horizontal. Hepatitis with basophilic intranuclear inclusion bodies distinguishes IBH, but the buildup of translucent or straw-colored fluid in the pericardial sac distinguishes HHS. IBH and HHS require a confirmatory diagnosis because their clinical symptoms and postmortem abnormalities are not unique to those conditions. Under a microscope, the presence of particular lesions and inclusion bodies may provide clues. Traditional virus isolation in avian tissue culture is more delicate than in avian embryonated eggs. Additionally, aviadenovirus may now be quickly and precisely detected using molecular diagnostic tools. Preventive techniques should rely on efficient biosecurity controls and immunize breeders prior to production in order to protect progeny. This current review gives a general overview of the current local and global scenario of IBH, and HHS brought on by FAdVs and covers both their issues and preventative vaccination methods.

MOLECULAR AND BIOLOGICAL CHARACTERIZATION OF FOWL AVIADENOVIRUS ISOLATED FROM COMMERCIAL CHICKEN FARMS

Fowl Adenovirus (FAdV) is a virus that causes disease in poultry with characteristics such as inclusionsbody found in organ lesions, especially the liver. The disease causes economic losses, in the form of chickendeaths and immunosuppression effects. Since 2018, outbreaks of FAdV or known as inclusion body hepatitis(IBH) have been reported in broiler farms, in several provinces in Indonesia. This study aims to isolate,and characterize molecularly, and biologically of embryonic chicken eggs and liver cell cultures of chickenembryos. Liver samples, bursa fabricius, and spleen were obtained from 23 commercial chicken farms inIndonesia that were diagnosed with IBH disease from 2018-2020. Molecular detection was carried out bypolymerase chain reaction (PCR) technique, using hexon gene-specific primers. The amplification resultwas positive, then sequencing was performed. Isolation was carried out using embryonic chicken eggs(ECE) specific pathogen free (SPF) and chicken embryo liver (CEL) cell cultures. Detection and molecularcharacterization showed that all FAdV isolated from poultry obtained from cases diagnosed with IBHdisease confirmed as FAdV genotype E, including strain FAdV-8b. Isolation on TAB showed embryonicbleeding lesions and swelling of the liver. Propagation of the FADV isolate using chicken embryo liver cellculture, showed cytopathic lesions that could be observed in the first passage, in the form of swelling androunding of infected cells. IBH disease outbreaks in commercial chickens in this study, confirmed the causeis FAdV genotype E, which is included in serotype 8b. FAdV virus isolated on SPF TAB showed embryonicbleeding lesions and hepatitis. FAdV virus isolated in this study was assessed as pathogenic in SPFchicken embryos.

Genetic evolution of fowl adenovirus serotype 4 and its pathogenicity to Cherry Valley ducks in China

Hydropericardium hepatitis syndrome (HHS), a novel poultry disease, is caused by fowl adenovirus 4 (FAdV-4). It mainly infects 3–5-week-old broilers. In July 2015, the first outbreak of HHS occurred in the broilers in east China, which caused great economic losses to the poultry industry. In June 2019, infectious disease was detected with suspected HHS symptoms on a duck farm in Linyi City, Shandong Province. The main necropsy lesions included pericardial effusion and hepatitis. In this study, we isolated a strain of FAdV-4 from naturally infected ducks and named it SDLY190604, and the hexon gene sequence was amplified and analyzed using polymerase chain reaction (PCR). In order to study the effect of FAdV-4 on Cherry Valley ducks, we inoculated three-week-old ducks with 0.2 ml of FAdV-4 virus fluid (TCID50 of 10-6.3/0.1 ml) by orally, subcutaneously and intramuscularly. Clinical signs, gross lesions and histopathological changes, cytokines and viral load were detected and recorded within 15 days after infection. The results showed that ducks in the experimental groups exhibited typical symptoms of hydropericardium and hepatitis. The histopathological sections showed multiple-organ damage, including serious liver and kidney damage with elevated levels of inflammatory cytokines, probably due to the infection and innate immune response. Later, immunosuppression occurred, resulting in decreased levels of cytokines. The viral load indicated that the virus could be present in several organs of the ducks, with the highest viral DNA found in the liver, followed by the kidney. Compared to the subcutaneous and oral groups, the intramuscular group exhibited the highest viral load. In summary, this study can increase our understanding of the pathogenicity of FAdV-4 in ducks and provide a basis for further understanding of the virus, imparting new insights into disease research.

Epidemiological investigation of infectious diseases in geese on mainland China during 2018-2021.

Geese play an important role in agricultural economics, with China producing the vast majority of goose meat consumed worldwide annually. The variations in the avian viruses and co-infections result in substantial economic losses to the goose industry in China. To understand the evolutionary characteristics and co-infections of viruses, a broad epidemiological investigation of epizootic viruses of goose was carried out in nine provinces of China during 2018-2021. Here, the results indicated that, among the 1970 clinical samples, 50.81% (1001/1970) were positive for goose astrovirus (GAstV), 18.22% (359/1970) for avian orthoreovirus, 12.74% (251/1970) for goose parvovirus, 11.02% (217/1970) for H9N2 subtype avian influenza virus, 4.01% (79/1970) for Newcastle disease virus, and 2.08% (41/1970) for fowl adenovirus. Among the six viruses, co-infections comprised a large proportion (66.37%) in the field, of which the dual infection was more common. Additionally, phylogenetic analysis of GAstVs indicated that Chinese GAstVs had formed two distinct groups, that is, GAstV-1 and GAstV-2. GAstV-2 sub-genotype II-c had arisen as the dominant genotype in the whole country. Notably, all the H9N2-AIV isolated strains harboured the mammalian adaptation markers I155T, H183N, and Q226L (H3 numbering) in the HA gene, which promotes preferential binding to human-like α2-6-linked sialic acid receptors. And beyond that, we determined that the goose-origin Muscovy Duck Reovirus isolates, showing 51.7%-96% similarities to that of other waterfowl-origin orthoreovirus isolates in sequence homology analysis of the representative part of σC, are a new variant of waterfowl-origin orthoreovirus. These data provide valuable information about the prevalence of infectious diseases in geese on mainland China.

Pathogenicity Assessment and Vaccine Efficacy of Fowl Adenovirus Serotype 4 and 11 Responsible for Inclusion Body Hepatitis Hydropericardium Syndrome in Broilers

Thisstudy aimed to assess the pathogenicity and vaccine efficacy of fowl adenovirus serotypes 4 and 11, causing inclusion body hepatitis hydropericardium syndrome in broilers. The study was carried out on 144-day-old Cobb broiler chicks divided into six experimental groups, each of 24 birds. Group, I birds served as a control without any vaccine or viral challenge, while birds of Group II were vaccinated with commercial vaccine on 6th day. Group III and IV birds were non-vaccinated and challenged with 107 TCID50 of FAdV serotype 4 and 11, respectively, on the 27th day. In contrast, Group V and VI birds were vaccinated on the 6th day and challenged with 107 TCID50 of FAdV serotype 4 and 11 on the 27th day. The challenge of FAdV serotype 4 led to 66.66% mortality, and serotype 11 produced 5.00% mortality in the experimental groups. The challenge of the virus led to the production of clinical signs such as depression, huddling, reduced feed intake, reluctance to move, and terminal gasping before death. Characteristic gross lesions of IBH-HPS were observed in birds that died after the challenge of serotypes 4 and 11 of FAdV. The liver was enlarged with focal areas of necrosis, subcapsular hemorrhages, and mild fatty changes. The heart was flabby, congested, and showed hydropericardium with an accumulation of clear watery to a yellow jelly-like fluid in the pericardial sac. The kidneys were enlarged and hemorrhagic. On microscopic examination, the liver showed multifocal areas of necrosis with infiltration of mononuclear cells, mild fatty changes and large basophilic intranuclear inclusion bodies. The birds that died after the challenge of the virus were positive for the presence of FAdV upon PCR. The vaccine efficacy study indicated that the vaccine provided satisfactory protection against Fowl Adenovirus challenge in experimental groups V and VI.

TMT-based quantitative proteomics analysis reveals the role of Notch signaling in FAdV-4-infected LMH cell.

Fowl adenovirus serotype 4 (FAdV-4) is recognized as a pathogen that causes hydropericardium syndrome. Irrespective of the pathway used by the virus to invade the chicken, the pathological characteristics of the disease include degeneration and necrosis of hepatocytes, formation of intranuclear inclusions, as well as inflammatory cell infiltration. Liver dysfunction constitutes one of the critical factors leading to death. Therefore, it is vital to investigate the virus-mediated severe pathological liver damage to further understand the pathogenesis of FAdV-4. Here, proteomics, a tandem mass tag (TMT)-based approach to directly analyze protein expression, was used to determine the protein expression during FAdV-4 proliferation in leghorn male hepatoma (LMH) cells. We identified 177 differentially expressed proteins associated with various biological processes and pathways. The functional enrichment analysis revealed that FAdV-4 could downregulate some signaling pathways in LMH cells, including NOD-like receptor signaling, RIG-I-like receptor signaling, NF-κB signaling, TNF signaling pathway, and Notch signaling, FoxO signaling, PI3K-Akt signaling, and autophagy. The results of proteomics screening suggested an association between FAdV-4 infection and Notch signaling in LMH in vitro, indicating that Notch signaling regulated the expression of inflammatory cytokines and interferons but not viral replication in LMH cells. These data contributed to the understanding of the immunopathogenesis and inflammopathogenesis of FAdV-4 infection and also provided valuable information for the further analysis of the molecular mechanisms underlying viral pathogenesis.

Genotyping and pathogenicity of fowl adenovirus isolated from broiler chickens in Egypt

BackgroundOver the past 10 years, inclusion body hepatitis outbreaks, essentially from commercial broiler flocks, have been detected in different geographic regions highlighting the wide distribution of FAdVs around the world resulting in serious economic losses due to increased mortalities as well as poor performance within poultry farms in Assiut province, Egypt. Thus, this study was achieved to detect fowl adenovirus in broiler chicken flocks in Assiut province, Egypt and to recognize the pathogenicity of the isolated virus.ResultsThe phylogeny of the L1 loop of the hexon gene exposed that the isolated virus clustered and belonged to the reference strains serotype D FAdV. The isolated virus is closely related to inclusion body hepatitis (IBH) strains causing extensive economic losses. The pathogenicity study of the virus showed typical macroscopic lesions with 6% mortality; furthermore, histopathological inspection exhibited severe hepatitis and degenerative changes after 5d from infection in the immune system. ConclusionResults in this research support the primary pathogenicity and mortality caused by FADV serotype 2 (IBH) alone without immunosuppressive agents thus robust control measures should be implanted against FAdV to evade the serious economic losses in poultry farms.

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.

Complete Genome Analysis and Animal Model Development of Fowl Adenovirus 8b.

Inclusion body hepatitis (IBH), hydropericardium syndrome, and gizzard erosion associated with fowl adenovirus (FAdV) infection have caused notable economic losses worldwide. In 2020, severe IBH was observed in a layer chicken farm in Hebei Province, China. Liver samples were collected from layer chickens with severe IBH and virus isolation was performed in LMH cells. DNA sequence and bioinformatics analyses were conducted to determine the phylogenetic relationship and the pathogenicity assay was conducted in specific-pathogen-free (SPF) chickens. HeB20 strain was isolated and identified as FAdV-8b, and the complete genome was successfully sequenced (GenBank No. OK188966). Although widespread recombination in clinical strains has been reported within FAdVs, HeB20 showed some novel characteristics, and did not show any recombination, highlighting that recombinant and non-recombinant FAdV-8b coexist in the clinic poultry industry. Finally, pathogenicity animal model of HeB20 was developed and showed severe IBH and 10% mortality. Collectively, a new FAdV-8b strain (HeB20) was isolated and responsible for the severe IBH in layer chickens. Complete genome of HeB20 was sequenced and valuable for future epidemiological investigations. HeB20 was capable of inducing severe IBH and 10% mortality in SPF chickens; this animal model provides a powerful tool for the future vaccine development.