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Severe hepatitis-hydropericardium syndrome (HHS) associated with a novel viral genotype, fowl adenovirus 4 (FAdV-4), has emerged and widely spread in China since 2015, causing severe economic losses to the poultry industry. We previously reported that the hexon gene is responsible for pathogenicity and obtained a non-pathogenic hexon-replacement rHN20 strain; however, the lack of information about the non-essential regions for virus replication limits the development of a FAdV-4 vector. This study first established an enhanced green fluorescent protein (EGFP)-indicator virus based on the FAdV-4 reverse genetic technique, effective for batch operations in the virus genome. Based on this, 10 open reading frames (ORFs) at the left end and 13 ORFs at the right end of the novel FAdV-4 genome were deleted separately and identified as non-essential genes for viral replication, providing preliminary insertion sites for foreign genes. To further improve its feasibility as a vaccine vector, seven combinations of ORFs were successfully replaced with EGFP without affecting the immunogenicity of the vector backbone. Finally, a recombinant rHN20-vvIBDV-VP2 strain, expressing the VP2 protein of very virulent infectious bursa disease virus (vvIBDV), was rescued and showed complete protection against FAdV-4 and vvIBDV. Thus, the novel FAdV-4 vector could provide sufficient protection for HHS and efficient exogenous gene delivery. Overall, our findings systemically identified 23 non-essential ORFs for FAdV-4 replication and seven foreign gene insertion regions, providing valuable information for an in-depth understanding of the novel FAdV-4 pathogenesis and development of multivalent vaccines. [ABSTRACT FROM AUTHOR] more...

• The rFH protein was expressed in E. coli system with good solubility. • Protection induced by different doses of rFH protein was assessed. • Antibody levels, clinical signs, tissue damages and viral load were evaluated. • The rFH subunit vaccine showed good protection against hyper-virulent virus. Hepatitis-hydropericardium syndrome (HHS) is widespread in China and causes high chicken mortality that results in great economic losses. A safe and effective vaccine is needed, and a subunit vaccine has potential for development. In this study, a truncated region of the FAdV-4 fiber 2 fused with coding sequence of one epitope of hexon was expressed in a prokaryotic expression system, and the immune protective effects of different doses of recombinant fiber 2 subunit vaccine on SPF chickens were compared. The recombinant fiber2 (Gly275- Pro479 aa)-hexon (Met21-Val51 aa) protein (rFH) obtained in Escherichia coli showed good solubility. The chicken survival rate at the lowest dose (2.5 μg/bird) was 75% (6/8), and at higher doses (≥5 μg/bird) was 100% (8/8) in challenge experiment. Two chickens in the 2.5 μg/bird treatment showed severe lesions, while birds in the higher dose treatments showed no obvious tissue damage as determined by histopathologic analysis of liver and spleen. Absolute quantitative real-time PCR showed no viral load in the ≥5 μg/bird treatments, but two chickens in the 2.5 μg/bird treatment had high viral loads. The challenge experience demonstrated that the rFH vaccine provided 100% protection at ≥5 μg/bird. These results suggested that rFH protein as an effective vaccine to protect against FAdV-4 and provided a new idea for the development of vaccine against HHS. [ABSTRACT FROM AUTHOR] more...

Hypervirulent fowl adenovirus serotype 4 (hvFAdV-4) has emerged as a major pathogen of hepatitis-hydropericardium syndrome (HHS) with increased mortality in chickens, resulting in economic losses to the Chinese poultry industry since June 2015. Here, we isolated a hypervirulent FAdV-4 (hvFAdV-4) strain (designated GD616) from 25-day-old meat-type chickens with severe HHS in Guangdong Province China in June 2017. The whole genome of the strain GD616 shares high homology with those in the recently-reported hvFAdV-4 isolates in China, with natural deletions of ORF19 and ORF27. A comparative analysis of Hexon and Fiber-2 proteins revealed that 2 unique amino acid residues at positions 378 and 453 of the Fiber-2 protein might be associated with virulence due to their occurrences in all the hvFAdV-4 isolates only. To systemically evaluate the effect of age on the susceptibility of chickens to hvFAdV-4, we used this hvFAdV-4 strain to intramuscularly inoculate 7- to 180-day-old specific-pathogen-free chickens for the evaluation of pathogenicity. These results showed that the pathogenicity of the hvFAdV-4 strain GD616 to chickens exhibited age-relatedness, with younger than 59-day-old chickens showing 100% morbidity and mortality, while 180-day-old chickens still exhibited a hydropericardium syndrome-like clinicopathology with 60% morbidity and 20% mortality. These findings enrich the current available knowledge regarding the pathogenicity of the hypervirulent FAdV-4 virus in chickens with a wide range of ages, which assists with the selection of suitable-aged chickens for the evaluation of hvFAdV-4 vaccines., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.) more...

Clinical cases of hepatitis-hydropericardium syndrome (HHS) from fowl adenovirus serotype 4 (FAdV-4) have increased in China since 2013. Therefore, the development of a new serologic method for HHS detection is now urgent. Here, the FAdV-4 strain JSJ13 was used to construct a plasmid for prokaryotic expression of the JSJ13 fiber-2 protein. The protein, purified by affinity chromatography, was refolded by gradient dialysis. After coating a 96-well plate with the purified fiber-2 protein (1.5 μg/ml), standard serum and secondary antibodies (1:200 and 1:6000 dilutions, respectively) were used to develop an indirect ELISA (I-ELISA). Nine field-collected serum samples and JSJ13-positive serum were tested by I-ELISA and the results corresponded with those of the serum neutralization test. The I-ELISA was used to test 450 clinical serum samples from different parts of China. Chickens from nonvaccinated flocks had low antibody titers and low virus positivity rates. In contrast, FAdV-4 vaccinated chickens were strongly positive, and the positivity rates of all the flocks exceeded 73.3%. The newly developed I-ELISA with the recombinant JSJ13 fiber-2 protein as the antigen detected antibodies to FAdV-4 accurately and sensitively. more...