Development of recombinant proteins for the control and diagnosis of Hydropericardium syndrome

Abstract

Hydropericardium syndrome (HPS) is a viral disease of poultry which is caused by Fowl adenovirus (FAdV). This virus belongs to family Adenoviridae and genus Aviadenovirus. In recent years, Hydropericardium syndrome (HPS) has emerged as one of the important diseases occurring in Pakistan and has caused heavy economic loss. Efforts have been made to develop conventional vaccines against this disease. These vaccines were formulated from infected liver homogenate. Unfortunately, formalin-inactivated liver organ vaccines failed to protect the poultry industry in the country. Hence, there is a need to develop a suitable vaccine to combat this disease. Currently, recombinant vaccine candidates are being developed for the prevention and control of some infectious diseases in several laboratories elsewhere. The present work is an effort to develop a recombinant protein, using molecular biology, biotechnological and immunological approaches for effective control and diagnosis of HPS. In the present study, the viral particle was isolated from natural outbreak of Hydropericardium syndrome in broilers, Punjab province of Pakistan using conventional methods. The existence of the virus was initially observed by Scanning Electron Microscopic examination. Icosahedral shaped viral particles of 70 – 80 nm in diameter were observed. Further, the presence of FAdV was confirmed by Polymerase Chain Reaction (PCR) by amplification of 730 bp variable region (L1 and part of P1 loop) of hexon gene. DNA sequence analysis and Phylogenetic analysis of the PCR product revealed that isolate is closely related with Indian fowl adenovirus – 4 isolate. To investigate which gene product encoded by fowl adenovirus plays vital role in immune response against the disease, two genes representing structural proteins of the virus (Penton base & short fiber) and one gene representing non structural protein (100K) were selected to develop recombinant constructs. To achieve this, the Penton base (1587bp), Short fiber (1437bp) and 100K (2397bp) genes were amplified by PCR and cloned in an expression vector (pET28a). The histidine residues along with thrombin protease site were engineered upstream to inserts (viral genes). The presence of recombinant DNA fragments were confirmed by double digestion method, PCR amplification of insert using gene specific primers and DNA sequencing of the inserts. Nucleotide sequences of inserts revealed that two genes (Penton base and Short fiber) of local isolate have >98% homology with the Indian FAdV-4 isolates, while one gene (100K) has 96% homology with the Russian FAdV-10 isolate. The recombinant constructs were expressed in E. coli. The expression of recombinant proteins was assessed by SDS-PAGE. Western blot analysis confirmed the presence of histidine tagged recombinant proteins i.e. short fiber (60Kda), penton base (65Kda) and 100K (95Kda) using anti His tag antibody. The three recombinant proteins were purified by Nickle affinity chromatography. The biological and immunological activity of recombinant proteins were assessed for potential use as antigen in vaccine and diagnostic (ELISA). The purified recombinant proteins were adjuvanted separately with Freund’s complete adjuvant and broilers were immunized. ELISA test was performed and antibody titers were determined against the respective recombinant proteins. The results indicated that protein constructs pSMJ-2 (penton base) and pSMJ-3 (short fiber) are more immunogenic antigens as compared to protein construct pSMJ-1 (100K) and commercial vaccine. Challenge protection test also proved that penton base (pSMJ-2) and short fiber (pSMJ- 3) protein constructs conferred 90% and 80% protection respectively against pathogenic virus challenge. Whereas 100K (pSMJ-1) protein construct and commercial inactivated vaccine provided 50% and 70% protection respectively. The results obtained by ELISA and challenge test in this study indicated that the constructed recombinant proteins are suitable candidates to develop subunit vaccine and diagnostic kit (strip test) thereby can be used for prevention and control of this disease.