Detection and differentiation of fowl adenovirus serotype 4 and duck adenovirus 3 using high resolution melting curve assay.

Fowl adenovirus type 4 (FAdV-4) and duck adenovirus type 3 (DAdV-3) are the causative agents of clinical diseases in poultry and have caused considerable economic losses to the waterfowl industry in China. Both FAdV-4 and DAdV-3 are classified into the genus Aviadenovirus under the family Adenoviridae. The high-resolution melting (HRM) assay has become a useful method for virus genotyping, which offers the possibility of rapidly developing a differentiation technique in which the melting profile depends on the GC content of the product in the qPCR platform. The aim of this study was to develop a qPCR-HRM assay for sensitive FAdV-4 and DAdV-3 detection and differentiation. Here, specific primers were designed on the basis of the 100 K genes of FAdV-4 and DAdV-3, and a qPCR-HRM assay was established through optimization of the reaction conditions. A specificity test revealed that this method could detect only FAdV-4 and DAdV-3, with no cross-reaction with other common duck-derived viruses. A sensitivity test revealed that the lowest detection limits of FAdV-4 and DAdV-3 were 2.84 copies/µL and 2.85 copies/µL, respectively. A repeatability test demonstrated that the coefficient of variation was less than 2.5 % in both the intragroup and the intergroup analyses. Field sample distributions of FAdV-4 and DAdV-3 were investigated, and the percentages of DAdV-3-positive, FAdV-4-positive and coinfection-positive in Muscovy ducks were 27.78 %, 16.67 % and 11.11 %, respectively. Further studies are needed to provide more insight into the pathogenesis of FAdV-4 and DAdV-3 coinfection in ducks. In conclusion, the qPCR-HRM assay provides an accurate, sensitive, reliable and cost-effective alternative method for detecting and distinguishing FAdV-4 and DAdV-3.

Precise location of three novel linear epitopes using the generated monoclonal antibodies against the Knob domain of FAdV-4 surface structural protein, fiber1.

Background: Fowl adenovirus serotype 4 (FAdV-4) is the main pathogen of hepatitis-hydropericardium syndrome (HHS), which brings huge economic losses to the poultry industry worldwide. Fiber-1 protein plays an important role in viral infection and pathogenesis by binding directly to cellular receptors of FAdV-4. In particular, the knob domain of fiber-1 protein has been reported to induce the production of neutralizing antibodies and arouse protection against the lethal challenge of chickens with FAdV-4. Methods: The fiber-1 knob (F1K) protein was expressed in a prokaryotic expression system and purified using Ni-NTA affinity chromatography. Monoclonal antibodies (mAbs) against FAdV-4 were generated by immunizing BALB/c mice with the purified F1K protein and screened using a series of immunoassays. Potential B cell epitopes on the knob domain of fiber-1 protein were mapped using enzyme-linked immunosorbent assay (ELISA) and dot-blot. Precious location and crucial amino acids of the identified epitopes were determined using peptide array scanning, truncations and alanine-scanning mutagenesis. The epitopes were analyzed and visualized on the knob trimer of FAdV-4 fiber-1 protein using the PyMOL software. Results: Water-soluble recombinant fiber-1 knob (F1K) protein was obtained with the assistance of chaperone. Four monoclonal antibodies (5C10, 6F8, 8D8, and 8E8) against FAdV-4 were generated and characterized using indirect ELISA, Western blot, dot-blot, and immunological fluorescence assay (IFA). The mAbs were demonstrated to be from different hybridoma cell lines based on the sequences of the variable regions. Meanwhile, three distinct novel linear B-cell epitopes (319SDVGYLGLPPH329, 328PHTRDNWYV336, and 407VTTGPIPFSYQ417) on the knob domain of fiber-1 protein were identified and the key amino acid residues in the epitopes were determined. Structural analysis showed that the two adjacent epitopes 319SDVGYLGLPPH329 and 328PHTRDNWYV336 were exposed on the surface of the fiber-1 knob trimer, whereas the epitope 407VTTGPIPFSYQ417 was located inside of the spatial structure. Conclusion: This was the first identification of B-cell epitopes on the knob domain of fiber-1 protein and these findings provided a sound basis for the development of subunit vaccines, therapeutics, and diagnostic methods to control FAdV infections.

Fowl adenovirus serotype 4 (FAdV-4) infection induces inflammatory responses in chicken embryonic cardiac fibroblasts via PI3K/Akt and IκBα/NF-κB signaling pathways.

Fowl adenovirus serotype 4 (FAdV-4) is the main pathogen of the acute infectious disease hepatitis-hydropericardium syndrome (HHS). Previous studies have focused on the mechanisms of FAdV-4 caused liver injury, while studies revealing potential mechanisms of inflammatory injury in FAdV-4-infected chicken cardiac cells remain scare. Here we found that FAdV-4 successfully infected chicken embryonic cardiac fibroblasts (CECF) cells in vitro and significantly upregulated production of inflammatory cytokines including IL-1ß, IL-6, IL-8, and TNF-α, suggesting induction of a strong inflammatory response. Mechanistically, FAdV-4 infection increased expression of phosphorylated Akt in a time-dependent manner, while phosphorylation of Akt and production of pro-inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α were greatly reduced in FAdV-4-infected CECF cells after treatment with LY294002, a potent inhibitor of PI3K, indicating that the inflammatory response induced by FAdV-4 infection is mediated by the PI3K/Akt signaling pathway. Furthermore, FAdV-4 infection increased expression of phosphorylated IκBα, a recognized indicator of NF-κB activation, and treatment with the BAY11-7082, a selective IκBα phosphorylation and NF-κB inhibitor, significantly reduced IκBα phosphorylation and inflammatory cytokines (IL-1ß, IL-6, IL-8, and TNF-α) production in FAdV-4-infected CECF cells, suggesting a critical role of IκBα/NF-κB signaling in FAdV-4-induced inflammatory responses in CECF cells. Taken together, our results suggest that FAdV-4 infection induces inflammatory responses through activation of PI3K/Akt and IκBα/NF-κB signaling pathways in CECF cells. These results reveal potential mechanisms of inflammatory damage in chicken cardiac cells caused by FAdV-4 infection, which sheds new insight into clarification of the pathogenic mechanism of FAdV-4 infection and development of new strategies for HHS prevention and control.

FAdV-4 can cause more noticeable clinical symptoms compared to FAdV-8b after infecting specific pathogen free chickens.

Chickens infected with FAdV-4 and FAdV-8b both exhibit hepatic lesions characterized by hemorrhagic necrosis and intranuclear inclusion body formation. However, only FAdV-4 induces pericardial effusion and acute mortality in chickens. To investigate the similarities and differences in the pathogenicity of HPS and IBH, this study intends to compare the infectivity and pathogenicity of FAdV-4 and FAdV-8b, 2 serotypes of fowl adenovirus isolated in our laboratory. The 2 viruses were respectively inoculated subcutaneously into SPF chicks at the neck. The clinical manifestations and pathological changes in these infected groups of chickens differed to some extent. Chickens infected with FAdV-4 exhibit evident depression and acute mortality, with a mortality rate of 60%; while those infected with FAdV-8b only display mild depression. Postmortem examination reveals serosanguinous effusion in the pericardial sac, spot-like hemorrhage, and focal necrosis in the liver of chickens infected with FAdV-4. Additionally, various degrees of edema are observed in organs such as the lungs, spleen, kidneys, and pancreas. In contrast, chickens infected with FAdV-8b exhibit spot-like hemorrhage and focal necrosis in the liver but do not display pericardial effusion or widespread organ edema. Histopathological examination demonstrates that both FAdV-4 and FAdV-8b can induce inflammatory reactions of varying degrees in the kidneys, pancreas, and duodenum of chickens, while reducing the necrosis of bursa of Fabricius, thymus, and spleen lymphocytes. Our data preliminarily reveal that both FAdV-4 and FAdV-8b can induce strong pathogenicity in chickens.

A novel subunit vaccine based on Fiber1/2 knob domain provides full protection against fowl adenovirus serotype 4 and induces stronger immune responses than a Fiber2 subunit vaccine.

Outbreaks of hepatitis-hydropericardium syndrome (HHS) caused by fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry in China since 2015. However, commercially available vaccines against the FAdV-4 infection remain scarce. In our study, subunit vaccine candidates derived from the bacterially expressed recombinant Fiber1 knob domain and Fiber2 knob domain fusion protein (termed as Fiber1/2 knob subunit vaccine) and Fiber2 protein (termed as Fiber2 subunit vaccine) of the FAdV-4 SDSX strain were developed. Immunogenicity evaluation showed that the Fiber1/2 knob subunit vaccine induced the production of antibodies at 7 d postvaccination (dpv), earlier than the Fiber2 subunit vaccine. Moreover, the neutralizing antibody level of the Fiber1/2 subunit vaccine group was higher than the Fiber2 subunit vaccine group, showing significant differences at 14, 21, and 28 dpv. Immune protection test results revealed that both Fiber1/2 knob subunit and Fiber2 subunit vaccines could protect chickens from death against FAdV-4 challenge, although the weight of chickens in the Fiber1/2 knob subunit vaccine group decreased less. Furthermore, analysis of plasma Glutamic oxaloacetic transaminase (AST) and blood glutamic pyruvic transaminase (ALT) levels suggested that the Fiber1/2 subunit vaccine can significantly inhibit liver damage caused by FAdV-4 infection and is more effective in blocking the pathogenicity of FAdV-4 in target organs. In addition, the Fiber1/2 knob subunit vaccine further reduced the viral load in different tissues and virus shedding in chickens than the Fiber2 subunit vaccine. Overall, the Fiber1/2 knob subunit vaccine was more effective than the Fiber2 subunit vaccine. These findings lay the foundation for the development of more effective FAdV-4 subunit vaccines.

Biological features of fowl adenovirus serotype-4.

Fowl adenovirus serotype 4 (FAdV-4) is highly pathogenic to broilers aged 3 to 5 weeks and has caused considerable economic loss in the poultry industry worldwide. FAdV-4 is the causative agent of hydropericardium-hepatitis syndrome (HHS) or hydropericardium syndrome (HPS). The virus targets mainly the liver, and HPS symptoms are observed in infected chickens. This disease was first reported in Pakistan but has now spread worldwide, and over time, various deletions in the FAdV genome and mutations in its major structural proteins have been detected. This review provides detailed information about FAdV-4 genome organization, physiological features, epidemiology, coinfection with other viruses, and host immune suppression. Moreover, we investigated the role and functions of important structural proteins in FAdV-4 pathogenesis. Finally, the potential regulatory effects of FAdV-4 infection on ncRNAs are also discussed.

FAdV-4-induced ferroptosis affects fat metabolism in LMH cells.

Ferroptosis is a form of controlled cell death that was first described relatively recently and that is dependent on the formation and accumulation of lipid free radicals through an iron-mediated mechanism. A growing body of evidence supports the close relationship between pathogenic infections and ferroptotic cell death, particularly for viral infections. Ferroptosis is also closely tied to the pathogenic development of hepatic steatosis and other forms of liver disease. Fowl adenovirus serotype 4 (FAdV-4) is a hepatotropic aviadenovirus causing hydropericardium syndrome (HPS) that is capable of impacting fat metabolism. However, it remains uncertain as to what role, if any, ferroptotic death plays in the context of FAdV-4 infection. Here, FAdV-4 was found to promote ferroptosis via the p53-SLC7A11-GPX4 axis, while ferrostain-1 was capable of inhibiting this FAdV-4-mediated ferroptotic death through marked reductions in lipid peroxidation. The incidence of FAdV-4-induced fatty liver was also found to be associated with the activation of ferroptotic activity. Together, these results offer novel insights regarding potential approaches to treating HPS.

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

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

Novel variant infectious bursal disease virus diminishes FAdV-4 vaccination and enhances pathogenicity of FAdV-4.

Infectious bursal disease virus (IBDV) caused an acute and highly contagious infectious disease characterized by severe immunosuppression, causing considerable economic losses to the poultry industry globally. Although this disease was well-controlled under the widely use of commercial vaccines in the past decades, the novel variant IBDV strains emerged recently because of the highly immunized-selection pressure in the field, posting new threats to poultry industry. Here, we reported novel variant IBDV is responsible for a disease outbreak, and assessed the epidemic and pathogenicity of IBDV in this study. Moreover, we constructed a challenge model using Fowl adenovirus serotype 4 (FAdV-4) to study on the immunosuppressive effect. Our findings underscore the importance of IBDV surveillance, and provide evidence for understanding the pathogenicity of IBDV.

The complete genome sequence of a new fowl adenovirus (Fadv-4) strain from a recent outbreak in a chicken farm in Iran.

The whole genomic sequence of fowl adenovirus C (FAdV-4) strain FAdV-4/Pasouk, isolated from chickens with hepatitis-hydropericardium syndrome (HHS) from an outbreak in Iran, has been deposited in GenBank under accession number ON652872. Notably, this FAdV-4 isolate exhibited significant genetic similarities to contemporary isolates originating from China, indicating a shared ancestry.

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.

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.

Prevalence and Molecular Characteristics of FAdV-4 from Indigenous Chicken Breeds in Yunnan Province, Southwestern China.

Fowl adenovirus-induced hepatitis-pericardial effusion syndrome outbreaks have been increasingly reported in China since 2015, resulting in substantial economic losses to the poultry industry. The genetic diversity of indigenous chicken results in different immune traits, affecting the evolution of these viruses. Although the molecular epidemiology of fowl adenovirus serotype 4 (FAdV-4) has been well studied in commercial broiler and layer chickens, the prevalence and genetic characteristics of FAdV-4 in indigenous chickens remain largely unknown. In this study, samples were collected from six indigenous chicken breeds in Yunnan province, China. FAdV-positive samples were identified in five of the six indigenous chicken populations via PCR and 10 isolates were obtained. All FAdVs belonged to serotype FAdV-4 and species FAdV-C. The hexon, fiber, and penton gene sequence comparison analysis demonstrated that the prevalence of FAdV-4 isolates in these chickens might have originated from other provinces that exported chicks and poultry products to Yunnan province. Moreover, several distinct amino acid mutations were firstly identified in the major structural proteins. Our findings highlighted the need to decrease inter-regional movements of live poultry to protect indigenous chicken genetic resources and that the immune traits of these indigenous chickens might result in new mutations of FAdV-4 strains.

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.

Rapid Construction of an Infectious Clone of Fowl Adenovirus Serotype 4 Isolate.

Adenovirus vectors possess a good safety profile, an extensive genome, a range of host cells, high viral yield, and the ability to elicit broad humoral and cellular immune responses. Adenovirus vectors are widely used in infectious disease research for future vaccine development and gene therapy. In this study, we obtained a fowl adenovirus serotype 4 (FAdV-4) isolate from sick chickens with hepatitis-hydropericardium syndrome (HHS) and conducted animal regression text to clarify biological pathology. We amplified the transfer vector and extracted viral genomic DNA from infected LMH cells, then recombined the mixtures via the Gibson assembly method in vitro and electroporated them into EZ10 competent cells to construct the FAdV-4 infectious clone. The infectious clones were successfully rescued in LMH cells within 15 days of transfection. The typical cytopathic effect (CPE) and propagation titer of FAdV-4 infectious clones were also similar to those for wild-type FAdV-4. To further construct the single-cycle adenovirus (SC-Ad) vector, we constructed SC-Ad vectors by deleting the gene for IIIa capsid cement protein. The FAdV4 infectious clone vector was introduced into the ccdB cm expression cassette to replace the IIIa gene using a λ-red homologous recombination technique, and then the ccdB cm expression cassette was excised by PmeI digestion and self-ligation to obtain the resulting plasmids as SC-Ad vectors.

Critical Role of Viral Protein Hexon in Hypervirulent Fowl Adenovirus Serotype-4-Induced Autophagy by Interaction with BAG3 and Promotion of Viral Replication in LMH Cells.

Hepatitis-pericardial syndrome (HHS) is an acute highly infectious avian disease caused by fowl adenovirus serotype 4 (FAdV-4), characterized by fulminant hepatitis and hydropericardium in broilers. Since 2015, a widespread epidemic has occurred in China due to the emergence of hypervirulent FAdV-4 (HPFAdV-4), causing huge losses to the stakeholders. However, the pathogenesis of HPFAdV-4 and the host responses to its infection remain elusive. Here, we show that infection of leghorn male hepatocellular (LMH) cells by HPFAdV-4 induced complete autophagy in cells and that the autophagy induced by recombinant HPFAdV-4-ON1 (rHPFAdV-4-ON1), a viral strain generated by replacing the hexon gene of wild-type HPFAdV-4 (HPFAdV-4-WT) with the one of nonpathogenic strain FAdV-4-ON1, was remarkably mitigated compared to that of the rHPFAdV-4-WT control, suggesting that HPFAdV-4 hexon is responsible for virus-induced autophagy. Importantly, we found that hexon interacted with a cellular protein, BAG3, a host protein that initiates autophagy, and that BAG3 expression increased in cells infected with HPFAdV-4. Furthermore, knockdown of BAG3 by RNA interference (RNAi) significantly inhibited HPFAdV-4- or hexon-induced autophagy and suppressed viral replication. On the contrary, expression of hexon markedly upregulated the expression of BAG3 via activating the P38 signaling pathway, triggering autophagy. Thus, these findings reveal that HPFAdV-4 hexon interacts with the host protein BAG3 and promotes BAG3 expression by activating P38 signaling pathway, thereby inducing autophagy and enhancing viral proliferation, which immensely furthers our understanding of the pathogenesis of HPFAdV-4 infection. IMPORTANCE HHS, mainly caused by HPFAdV-4, has caused large economic losses to the stakeholders in recent years. Infection of leghorn male hepatocellular (LMH) cells by HPFAdV-4 induced complete autophagy that is essential for HPFAdV-4 replication. By a screening strategy, the viral protein hexon was found responsible for virus-induced autophagy in cells. Importantly, hexon was identified as a factor promoting viral replication by interaction with BAG3, an initiator of host cell autophagy. These findings will help us to better understand the host response to HPFAdV-4 infection, providing a novel insight into the pathogenesis of HPFAdV-4 infection.

Design and Identification of a Novel Antiviral Affinity Peptide against Fowl Adenovirus Serotype 4 (FAdV-4) by Targeting Fiber2 Protein.

Outbreaks of hydropericardium hepatitis syndrome caused by fowl adenovirus serotype 4 (FAdV-4) with a novel genotype have been reported in China since 2015, with significant economic losses to the poultry industry. Fiber2 is one of the important structural proteins on FAdV-4 virions. In this study, the C-terminal knob domain of the FAdV-4 Fiber2 protein was expressed and purified, and its trimer structure (PDB ID: 7W83) was determined for the first time. A series of affinity peptides targeting the knob domain of the Fiber2 protein were designed and synthesized on the basis of the crystal structure using computer virtual screening technology. A total of eight peptides were screened using an immunoperoxidase monolayer assay and RT-qPCR, and they exhibited strong binding affinities to the knob domain of the FAdV-4 Fiber2 protein in a surface plasmon resonance assay. Treatment with peptide number 15 (P15; WWHEKE) at different concentrations (10, 25, and 50 µM) significantly reduced the expression level of the Fiber2 protein and the viral titer during FAdV-4 infection. P15 was found to be an optimal peptide with antiviral activity against FAdV-4 in vitro with no cytotoxic effect on LMH cells up to 200 µM. This study led to the identification of a class of affinity peptides designed using computer virtual screening technology that targeted the knob domain of the FAdV-4 Fiber2 protein and may be developed as a novel potential and effective antiviral strategy in the prevention and control of FAdV-4.

Complete genome sequence and pathogenicity analysis of a highly pathogenic FAdV-4 strain.

Fowl adenovirus serotype 4 (FAdV-4) is a double-stranded DNA virus that mainly infects broiler chickens and has caused huge economic losses to the poultry industry. Recently, an FAdV-4 strain, SDLC202009, the causative pathogen of hydropericardium-hepatitis syndrome (HHS) in Liaocheng, Shandong, was isolated from commercial laying hens and propagated in specific pathogen free SPF chicken embryos. Pathogenicity studies showed that SDLC202009 could infect SPF chicken embryos and chickens, with a mortality rate of 100%. The complete genome was sequenced, and phylogenetic analysis showed that SDLC202009 belonged to the FAdV-4 cluster, with a genome length of 43, 077 bp. The SDLC202009 had 99.9% identity with the JSJ13 and SD1601, which were recently isolated in China. Compared to the recently isolated strain in China, SDLC202009 had deleted open reading frame 19 (ORF19), ORF27, ORF48, and ORF0. SDLC202009 harbored amino acid site mutations in the main structural proteins hexon, fiber1, and fiber2 similar with those in highly pathogenic strains. Furthermore, SDLC202009 showed unique mutations in hexon A571P, fiber1 E216K, and fiber2 N98K. In summary, our findings provide theoretical support for prevention and control of the HHS.

Knob domain of Fiber 2 protein provides full protection against fowl adenovirus serotype 4.

Highly pathogenic fowl adenovirus serotype 4 (FAdV-4) is an acute infectious disease with severe economic impact, causing chicken hepatitis hydropericardium syndrome (HHS) and high mortality. In the present study, we evaluated the immunogenicity of the recombinant Fiber2-knob protein (F2-Knob) as an FAdV-4 candidate subunit vaccine in 14-day-old SPF chickens. The knob domain is the functional region of the viral surface protein Fiber2. The protein was expressed in Escherichia coli and was administered a single immunization with different vaccine doses. The protective efficacy was evaluated by mortality, clinical symptoms, virus shedding and histopathological examinations after challenged with the FAdV-4. The results showed that the level of ELISA antibodies of the chickens immunized with Fiber2-knob protein was significantly higher than that of the chickens immunized with an inactivated vaccine against FAdV-4. The antibody value of the immunized Fiber2-knob protein was positively correlated with the increase in immunization dose. The challenge experiment showed that the F2-Knob protein provided full protection against virulent FAdV-4 challenge and significantly reduced viral shedding. These results suggest that F2-Knob protein could be a novel vaccine candidate provide insights to control FAdV-4.