Hydropericardium syndrome, a disease primarily of broiler birds, has emerged as a severe hazard to poultry producers, particularly in India and Pakistan and has led to closure of many farms. It causes high morbidity and variable mortality (Shane, 1996). However, the disease caused heavy mortality of 20-75% in Pakistan (Cheemaet al., 1989) and 30-80% in India, with no apparent prior signs of disease (Ravi Kumaret al., 1997). A group I fowl adenovirus, serotype 4 (Jadhaoet al., 1997; Chandraet al., 1997b), has been identified as the causative agent of Hydropericardium hepatitis syndrome (HHS).

The Fowl adenovirus 4 can be grown in primary cell cultures of chicken kidney (Khawajaet al., 1988a) or chicken embryonic liver cells (Naeemet al., 1995; Oberoiet al., 1996; Jadhaoet al., 1997). Many workers have purified the adenovirus using either sucrose, cesium chloride (CsCl₂) or potassium tartrate linear density gradient ultracentrifugation (Zsak and Kisary, 1984; Mendelsonet al., 1995; Ernyet al., 1995; Mazaheriet al., 1998; Balamurugan, 1999). However, these methods are tedious and time consuming for routine procedures. Capuaet al. (1995) purified the adenovirus associated with inclusion body hepatitis in psittacine birds by homogenizing the CEL cell propagated virus with chloroform and pelleting the virus at 130,000 g for 3 h. In the present study, an Indian isolate of fowl adenovirus 4 was propagated in primary chick embryo liver cell culture and the virus was purified by ultracentrifugation.

Twelve days old embryonated eggs were obtained from the Experimental hatchery unit of the Central Avian Research Institute, Izatnagar, Bareilly for the preparation of Chick embryo liver cell culture. One day old broiler chicks from apparently healthy parent stock were obtained from the Experimental hatchery unit of CARI, Izatnagar and were maintained at the Experimental Animal sheds of the Division of Standardization during the course of the study for initial passage of the virus. The Fowl adenovirus serotype 4 used in the present study was obtained from the Poultry Vaccine Testing Laboratory of the Division of Biological Standardization, Indian Veterinary Research Institute, Izatnagar, Bareilly. Fowl adenovirus serotype 4 obtained in the form of lyophilized liver homogenate was used to infect apparently healthy day old broiler chicks. Liver from chicks following death after 4-5 days postinfection showing the pathognomonic lesions such as hydropericardium and necrotic hepatitis were collected, homogenised in cold and clarified through centrifugation to prepare a 20% liver homogenate (w/v)in phosphate buffered saline (0.01 M pH 7.4). Antibiotics (Penicillin 1000 IU/ml and Streptomycin 1mg/ml) were added and filtered through 0.22 μm millipore filter. The liver homogenate was used as inoculum for infecting primary chick embryo liver cell culture.

Primary chick embryo liver cell culture was prepared as per the method of Adair et al. (1979) from 12–14 days old embryonated chicken eggs. Liver from the embryos were removed aseptically and washed twice with Phosphate buffered saline (pH 7.4). The washed livers were finely minced with curved scissors. The minced pieces were washed thrice with PBS and trypsinized gently using trypsin-versene solution (trypsin 0.17% & versene 0.14%)) at 37°C for few minutes. The trypsinized liver suspension was then filtered through a muslin cloth in a beaker. The collected filtrate was centrifuged at 2000 rpm for 10 min and the cell pellet obtained was washed twice with DMEM supplemented with 15% (v/v) newborn calf serum (NBCS) to remove the residual trypsin. The final cell pellet was resuspended in DMEM containing 15% (v/v) New born calf serum (NBCS) and the cell concentration was adjusted to 3 × 10⁶ cells/ml. The resuspended CEL cells were dispensed into 75 cm² and 175 cm² plastic tissue culture flasks and incubated at 37°C for 48–72 h until a uniform monolayer of the chicken embryo liver cells was formed.

Flasks containing complete monolayers were washed twice with serum-free DMEM and infected with 1 ml of virus per 75 cm² flask area. The virus inoculum was allowed to adsorb onto the cells at 37°C for 30 min. Following adsorption, maintenance medium containing 2% NBCS was added to the culture and the infected flasks were incubated at 37°C and the monolayer was observed for CPE daily for about four days. CEL cultures were harvested when the monolayers exhibited more than 80% cytopathic effects. The virus was passaged six times in chick embryo liver cell culture. The CEL cell culture was prepared in 175 cm² flasks (Griener, Germany) for the bulk production of the virus and the flasks were stored at −20°C for subsequent purification and extraction of DNA.

The fowl adenovirus serotype 4 was concentrated and purified following the method described by Capuaet al. (1995) and Jadhaoet al. (2000). CEL cell culture propagated virus was frozen and thawed three times. The cells were removed from the surface of the plastic flasks using cell scraper and the culture supernatant was pooled. The total volume of the culture supernatant was homogenized with 50% (v/v) reagent grade chloroform using dounce homogenizer. The homogenized suspension was centrifuged at 6000 g for 30 min at 4°C in a high-speed centrifuge. The pellet along with the chloroform was discarded and the supernatant was collected and centrifuged at 1,30,000 g for 2 h at 4°C in A-641 fixed angle rotor (OTD 75 D Sorvall, Ultracentrifuge, USA). The resultant virus pellet was suspended in 0.5 ml TE buffer (100 mM Tris HCL pH 8.0, 10mM EDTA pH 8.0) per tube and kept at 4°C overnight to dissolve the pellet completely. Subsequently, the virus pellet was then layered onto 3.0 ml cushion of 30% (w/v) sucrose and centrifuged at 1,00,000 g for 2 h at 4°C in swing out rotor. The resulting virus pellet was dissolved in 0.5 ml TE buffer (100 mM Tris HCL pH 8.0, 10mM EDTA pH 8.0) and collected in sterile microcentrifuge tubes and stored at −20°C until further processing for extraction of DNA. Optical density of the purified virus was taken at 260 nm and 280 nm in the spectrophotometer. The protein of the sample was determined using the formula: Protein (mg/ml) = OD₂₆₀ × 1.55–OD₂₈₀ × 0.77. Ratio of absorbance values at 260/280 nm was calculated to check the purity of the virus sample. The virus preparation having an OD of 1.2 was considered as purified.

Nested PCR was done to amplify 447 bp product from cell culture virus using the primers, Forward 5′-CTCACTGCGGCACGGATTACAACC-3′ (24 mer) and reverse 5′-CGGGGAGGCGGACGACTATG-3′ (20 mer). The reaction mixture consisted of 10 x PCR buffer 5 μl, 25 mM MgCl₂ 3 μl, Q-solution 10 μl, 10 mM dNTP mix 1 μl, Forward primer 2 μl, Reverse Primer 2 μl, FAV4 DNA (100 ng) 1 μl, Taq DNA polyerase 1 μl, DEPC water 27 μl. The cyclic conditions were initial denaturation 94°C for 2 min, 35 cycles of denaturation 94°C for 20 sec, annealing 63°C for 1 min, extension 72°C for 10 min and the final extension at 72°C for 10 min.

The fowl adenovirus serotype 4 (FAV4) produced CPE from the 3rd passage onwards in chick embryo liver cell culture. The virus passage was done six times in CEL cell culture. The CPE in infected CEL cell cultures appeared within 48 h which was characterised by swelling and rounding of the infected cells. By 72 h post infection, the cells became refractile and started detaching from the surface. At 96 h of infection, the monolayers completely detached from the surface. The healthy uninfected CEL cell cultures, however did not show any changes. Upon subsequent passages, the CPE started appearing early within 24 to 48 h and became more pronounced involving most of the cells in monolayer. The infectivity titre of the virus at the 6th passage level was 10^5.4 TCID50/ml. In the present study, the FAV4 was propagated in primary chick embryo liver cell culture which has a greater sensitivity than the kidney cultures (Swainet al., 1993). The virus produced rounding, degeneration and detachment of the infected cells in the monolayer, which corroborates the observation of Adairet al. (1979). Earlier workers had grown FAV4 in primary cell cultures of chicken kidney (Khawaja et al., 1989) or chick embryo liver cells (Naeemet al., 1995, Oberoiet al., 1996; Jadhaoet al., 1997). The cell culture as template fluid was used to amplify 447 bp FAV4 DNA product and this confirmed the virus in infected cell culture fluid. The infected cell culture fluid when inoculated into chicks by intramuscular route, produced disease in the chicks which ultimately died showing typical lesions of the disease in heart.

The fowl adenovirus serotype 4, purified by ultracentrifugation had a protein concentration of 3.56 mg/ml and the extinction ratio (OD₂₆₀/OD₂₈₀) of the purified virus was 1.14. The present study is in agreement of the observations of Capuaet al. (1995) and Jadhaoet al. (2000) who suggested that pelleting the virus by ultracentrifugation could be used for routine purification protocols. The protein concentration of the purified virus prepared by ultracentrifugation from one litre culture was 3.56 mg/ml while the extinction ratio of 1.14 indicated satisfactory purity of the virus preparation.

References