Breaking the Chain: Strategies to Stem Adenovirus Spread in Pakistan.

Adenovirus, a common respiratory pathogen, has witnessed a notable rise in incidence rates across various regions in Pakistan. Utilizing epidemiological data and climate records, this research discerns a potential linkage between the burgeoning adenovirus cases and alterations in regional climate patterns. Through statistical analysis and modeling techniques, the study aims to elucidate the relationship between climatic variables, such as temperature, humidity, and precipitation, and the prevalence of adenovirus infections. Understanding these dynamics is crucial for developing effective public health interventions and preparedness strategies to mitigate the impact of adenovirus outbreaks in Pakistan. Furthermore, this research contributes to the broader discourse on the intersection of infectious diseases and climate change, highlighting the need for comprehensive adaptive measures to address emerging health challenges in a changing environment.

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.

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.

Rotavirus and adenovirus infections in children with acute gastroenteritis after introducing the Rotasiil® vaccine in Kisangani, Democratic Republic of the Congo.

BACKGROUND: Although rotavirus vaccination has reduced the global burden of the virus, morbidity and mortality from rotavirus infection remain high in Sub-Saharan Africa. This study aimed to determine the prevalence of rotavirus and adenovirus infections in children under five years with acute gastroenteritis and to identify factors associated with rotavirus infection after the introduction of the Rotasiil® vaccine in 2019 in Kisangani, Democratic Republic of the Congo (DRC). METHODS: This study consisted of a cross-sectional hospital-based survey conducted from May 2022 to April 2023 in four health facilities in Kisangani, using a fecal-based test (rapid antigenic immuno-chromatographic diagnostic test, BYOSYNEX adenovirus/rotavirus BSS, Biosynex SA, Illkirch-Graffenstaden, France) of rotavirus and adenovirus infections among children under five years of age with acute gastroenteritis. RESULTS: A total of 320 children under five years of age with acute gastroenteritis were included. The prevalence of rotavirus infection was 34.4%, that of adenovirus was 6.3%, and that of both rotavirus and adenovirus coinfection was 1.3%. The prevalence of rotavirus was significantly higher in unvaccinated children than in vaccinated children (55.4% versus 23.1%; P < 0.001). This difference was observed only in children who received all three vaccine doses. Multivariate logistic regression analysis shows that the rate of rotavirus infection was significantly reduced in vaccinated children (adjusted OR: 0.31 [95% confidence intervals (CI): 0.19-0.56]; P < 0.001) and those whose mothers had an average (adjusted OR: 0.51 [95% CI: 0.25-0.91]; P = 0.018) or high level (adjusted OR: 0.34 [95% CI: 0.20-0.64]; P < 0.001) of knowledge about the rotavirus vaccine. CONCLUSIONS: The prevalence of rotavirus infection remains high in Kisangani despite vaccination. However, the prevalence of adenovirus infections was low in our series. Complete vaccination with three doses and mothers' average and high level of knowledge about the rotavirus vaccine significantly reduces the rate of rotavirus infection. It is, therefore, essential to strengthen the mothers' health education, continue with the Rotasiil® vaccine, and ensure epidemiological surveillance of rotavirus infection.

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.

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

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

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.

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

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

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

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

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.

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.

An inactivated novel chimeric FAdV-4 containing fiber of FAdV-8b provides full protection against hepatitis-hydropericardium syndrome and inclusion body hepatitis.

Fowl adenovirus serotype 4 (FAdV-4) and FAdV-8b are causative agents of hepatitis-hydropericardium syndrome (HHS) and inclusion body hepatitis (IBH), respectively. HHS and IBH co-infections were often reported in clinical, yet there are no commercially available bivalent vaccines for prevention and control of both FAdV-4 and -8b. In the present study, a chimeric FAdV-4 was firstly generated by substituting fiber-1 of FAdV-4 with fiber of FAdV-8b. The chimeric virus, rFAdV-4-fiber/8b, exhibited similar replication ability in vitro and pathogenicity in vivo to the parental wild type FAdV-4. A single dosage of vaccination with the inactivated rFAdV-4-fiber/8b induced high antibody titers against fiber-2 of FAdV-4 and fiber of FAdV-8b and provided full protection against FAdV-4 and -8b challenge. These results demonstrated that fiber of FAdV-8b could replace the role of fiber-1 of FAdV-4 in the process of viral infection, and rFAdV-4-fiber/8b could be used to make a potential bivalent vaccine for the control and prevention of HHS and IBH.

Probiotics Surface-Delivering Fiber2 Protein of Fowl Adenovirus 4 Stimulate Protective Immunity Against Hepatitis-Hydropericardium Syndrome in Chickens.

Background and Objectives: Hepatitis-hydropericardium syndrome (HHS) caused by Fowl adenoviruses serotype 4 (FAdV-4) leads to severe economic losses to the poultry industry. Although various vaccines are available, vaccines that effectively stimulate intestinal mucosal immunity are still deficient. In the present study, novel probiotics that surface-deliver Fiber2 protein, the major virulence determiner and efficient immunogen for FAdV-4, were explored to prevent this fecal-oral-transmitted virus, and the induced protective immunity was evaluated after oral immunization. Methods: The probiotic Enterococcus faecalis strain MDXEF-1 and Lactococcus lactis NZ9000 were used as host strains to deliver surface-anchoring Fiber2 protein of FAdV-4. Then the constructed live recombinant bacteria were orally vaccinated thrice with chickens at intervals of 2 weeks. Following each immunization, immunoglobulin G (IgG) in sera, secretory immunoglobulin A (sIgA) in jejunum lavage, immune-related cytokines, and T-cell proliferation were detected. Following challenge with the highly virulent FAdV-4, the protective effects of the probiotics surface-delivering Fiber2 protein were evaluated by verifying inflammatory factors, viral load, liver function, and survival rate. Results: The results demonstrated that probiotics surface-delivering Fiber2 protein stimulated humoral and intestinal mucosal immune responses in chickens, shown by high levels of sIgA and IgG antibodies, substantial rise in mRNA levels of cytokines, increased proliferative ability of T cells in peripheral blood, improved liver function, and reduced viral load in liver. Accordingly, adequate protection against homologous challenges and a significant increase in the overall survival rate were observed. Notably, chickens orally immunized with E. faecalis/DCpep-Fiber2-CWA were completely protected from the FAdV-4 challenge, which is better than L. lactis/DCpep-Fiber2-CWA. Conclusion: The recombinant probiotics surface-expressing Fiber2 protein could evoke remarkable humoral and cellular immune responses, relieve injury, and functionally damage target organs. The current study indicates a promising method used for preventing FAdV-4 infection in chickens.

Oral immunization of recombinant Saccharomyces cerevisiae expressing fiber-2 of fowl adenovirus serotype 4 induces protective immunity against homologous infection.

Hydropericardium-hepatitis syndrome (HHS) caused by fowl adenovirus (FAdV) serotype 4 strains is a highly contagious disease that causes significant economic loss to the global poultry industry. However, subunit vaccine against FAdV-4 infection is not yet commercially available to date. This study aims to explore the potential for oral immunization of recombinant Saccharomyces cerevisiae expressing Fiber-2 of FAdV-4 as a subunit vaccine. Here, we constructed recombinant S. cerevisiae (ST1814G/Fiber-2) expressing recombinant Fiber-2 (rFiber-2), which was displayed on the cell surface. To evaluate the immune response and protective effect of live recombinant S. cerevisiae, chickens were orally immunized with the constructed live ST1814G/Fiber-2, three times at 5-day intervals, and then challenged with FAdV-4. The results showed that oral administration of live ST1814G/Fiber-2 could stimulate the production of humoral immunity, enhance the body's antiviral activity and immune regulation ability, improve the composition of gut microbiota, provide protection against FAdV-4 challenge, reduce viral load in the liver, and alleviate the pathological damage of heart, liver, and spleen for chicken. In addition, we found the synergistic effect in combining the ST1814G/Fiber-2 yeast and inactivated vaccine to trigger stronger humoral immunity and mucosal immunity. Our results suggest that oral live ST1814G/Fiber-2 is a potentially safer auxiliary preparation strategy in controlling FAdV-4 infection.

Advances in Vaccine Development of the Emerging Novel Genotype Fowl Adenovirus 4.

Fowl adenovirus (FAdV) was first reported in Angara Goth, Pakistan, in 1987. For this reason, it is also known as "Angara disease." It was later reported in China, Japan, South Korea, India, the United States, Canada, and other countries and regions, causing huge economic losses in the poultry industry worldwide. Notably, since June 2015, a natural outbreak of severe hydropericardium hepatitis syndrome (HHS), associated with a hypervirulent novel genotype FAdV-4 infection, has emerged in most provinces of China. The novel virus FAdV-4 spread rapidly and induced a 30-100% mortality rate, causing huge economic losses and threatening the green and healthy poultry breeding industry. Vaccines against FAdV-4, especially the emerging novel genotype, play a critical role and will be the most efficient tool for preventing and controlling HHS. Various types of FAdV-4 vaccines have been developed and evaluated, such as inactivated, live-attenuated, subunit, and combined vaccines. They have made great contributions to the control of HHS, but the details of cross-protection within FAdVs and the immunogenicity of different vaccines require further investigation. This review highlights the recent advances in developing the FAdV-4 vaccine and promising new vaccines for future research.

Identification of Inhibitors and Drug Targets for Human Adenovirus Infections.

Adenoviruses can cause infections in people of all ages at all seasons of the year. Adenovirus infections cause mild to severe illnesses. Children, immunocompromised patients, or those with existing respiratory or cardiac disease are at higher risk. Unfortunately, there are no commercial drugs or vaccines available on the market for adenovirus infections. Therefore, there is an urgent need to discover new antiviral drugs or drug targets for adenovirus infections. To identify potential antiviral agents for adenovirus infections, we screened a drug library containing 2138 compounds, most of which are drugs with known targets and past phase I clinical trials. On a cell-based assay, we identified 131 hits that inhibit adenoviruses type 3 and 5. A secondary screen confirmed the antiviral effects of 59 inhibitors that inhibit the replication of adenoviruses type 3 or 5. Most of the inhibitors target heat shock protein, protein tyrosine kinase, the mTOR signaling pathway, and other host factors, suggesting that these host factors may be essential for replicating adenoviruses. Through this study, the newly identified adenovirus inhibitors may provide a start point for developing new antiviral drugs to treat adenovirus infections. Further validation of the identified drug targets can help the development of new therapeutics against adenovirus infections.

FAdV-4 without Fiber-2 Is a Highly Attenuated and Protective Vaccine Candidate.

Hepatitis-hydropericardium syndrome (HHS) caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4) has resulted in huge economic losses to the poultry industry globally. The fiber-2 gene, as a major virulence determiner, is also an important vaccine target against FAdV-4. In this study, we used a CRISPR/Cas9-based homology-dependent recombinant technique to replace the fiber-2 gene with egfp and generate a novel recombinant virus, designated FAdV4-EGFP-rF2. Although FAdV4-EGFP-rF2 showed low replication ability compared to the wild-type FAdV-4 in LMH cells, FAdV4-EGFP-rF2 could effectively replicate in LMH-F2 cells with the expression of Fiber-2. Moreover, FAdV4-EGFP-rF2 was not only highly attenuated in chickens, but also could provide efficient protection against a lethal challenge of FAdV-4. Moreover, FAdV4-EGFP-rF2 without fiber-2 could induce neutralizing antibodies at the same level as FA4-EGFP with fiber-2. These results clearly demonstrate that although fiber-2 affects the viral replication and pathogenesis of FAdV-4, it is not necessary for virus replication and induction of neutralizing antibodies; these findings provide novel insights into the roles of fiber-2 and highlight fiber-2 as an insertion site for generating live-attenuated FAdV-4 vaccines against FAdV-4 and other pathogens. IMPORTANCE Among all serotypes of fowl adenovirus, serotypes FAdV-1, FAdV-4, and FAdV-10 are unique members with two fiber genes (fiber-1 and fiber-2). Recent studies reveal that Fiber-1, not Fiber-2, directly triggers viral infection of FAdV-4, whereas Fiber-2, but not Fiber-1, has been identified as the major virulence determiner and an efficient protective immunogen for subunit vaccines. Here, we replaced fiber-2 with egfp to generate a novel recombinant virus, designated FAdV4-EGFP-rF2. In vitro and in vivo studies on FAdV4-EGFP-rF2 revealed that fiber-2 was not necessary for either virus replication or efficient protection for FAdV-4; these results not only provide a novel live-attenuated vaccine candidate against HHS, but also give new ideas for generating a FAdV-4 based vaccine vector against other pathogens.

Vaccination with a fowl adenovirus chimeric fiber protein (crecFib-4/11) simultaneously protects chickens against hepatitis-hydropericardium syndrome (HHS) and inclusion body hepatitis (IBH).

In the past decades, fowl adenovirus (FAdV)-related diseases became an increasing concern for the poultry industry worldwide. Various immunization strategies against FAdVs have been experimentally investigated, with a particular focus on subunit vaccines against hepatitis-hydropericardium syndrome (HHS), caused by FAdV serotype 4, and inclusion body hepatitis (IBH), caused by serotypes 2, 8a, 8b and 11. In this study, we extended our innovative concept of recombinant chimeric fiber proteins to design a novel chimera combining epitopes from two distinct serotypes, FAdV-4 and -11, and we investigated its efficacy to simultaneously protect chickens against HHS and IBH. Specific pathogen-free chickens were vaccinated with the novel recombinant chimeric fiber and subsequently challenged with either a HHS- or IBH-causing strain. Vaccinated/challenged birds exhibited a reduction of clinical signs, limited hepatomegaly and lower levels of AST compared to the respective challenge controls. Furthermore, the vaccine prevented atrophy of HHS-affected lymphoid organs, such as thymus and bursa of Fabricius, and viral load in the target organs was significantly reduced. Clinical protection was associated with high levels of pre-challenge antibodies measured on ELISA plates coated with the vaccination antigen. Interestingly, the development of neutralizing antibodies was limited against FAdV-11 and absent against FAdV-4, indicating that protection granted by such an antigen may be linked to different immunization pathways. In conclusion, we proved that the concept of chimeric fiber vaccines can be extended across viral species boundaries and represents the first single-component FAdV subunit vaccine providing comprehensive protection against different FAdV-associated diseases.

A Novel Recombinant FAdV-4 Virus with Fiber of FAdV-8b Provides Efficient Protection against Both FAdV-4 and FAdV-8b.

Since 2015, the outbreaks of hydropericardium-hepatitis syndrome (HHS) and inclusion body hepatitis (IBH) caused by the highly pathogenic serotype 4 fowl adenovirus (FAdV-4) and serotype 8 fowl adenovirus (FAdV-8), respectively, have caused huge economic losses to the poultry industry. Although several vaccines have been developed to control HHS or IBH, a recombinant genetic engineering vaccine against both FAdV-4 and FAdV-8 has not been reported. In this study, recombinant FAdV-4 expressing the fiber of FAdV-8b, designated as FA4-F8b, expressing fiber of FAdV-8b was generated by the CRISPR-Cas9 and homologous recombinant techniques. Infection studies in vitro and in vivo revealed that the FA4-F8b replicated efficiently in LMH cells and was also highly pathogenic to 2-week-old SPF chickens. Moreover, the inoculation of inactivated the FA4-F8b in chickens could not only induce highly neutralizing antibodies, but also provide efficient protection against both FAdV-4 and FAdV-8b. All these demonstrate that the inactivated recombinant FA4-F8b generated here can act as a vaccine candidate to control HHS and IBH, and FAdV-4 can be an efficient vaccine vector to deliver foreign antigens.