Molecular charecterization of Avian Adeno virus causing Inclusion Body Hepatitis-Hydropericardium syndrome in broiler

Avian Adenovirus was isolated from naturally infected broiler chickens. Two Liversample were collected in glycerol saline from the birds came from Aman and Jankipoutry farm for the postmortem in the Dept. of pathology,Veterinary college,Anand(Gujarat). Extraction of viral DNA from infected liver tissues was done as per the method of Meuleman et al., (2001) with minor modifications. The amplified PCR analyzed by agarose gel electrophoresis indicated DNA fragments of approximately 890 bp as expected by primer HexonA F & HexonB R. PCR assay revealed presence of IBH-HPS virus in both samples. Obtained PCR product of both sample were subjected to DNA sequencing and obtained sequencing was compared with other matched sequince. On phylogenetic analysis using Clustal W program showed 3 major group like upper, middle and lower respectively. In the minor branch of upper group the AMAN and JANKI isolates were found to group with Fowl adenovirus 12 strain 380 and Fowl adenovirus 11 strain C2B, so AMAN and JANKI isolates indicating a new fowl adenovirus genotype.


Introduction
is associated with the interpretation of results, as antibodies against adenoviruses are commonly The avian adenovirus infections are believed found in the blood of both healthy and infected to cause heavy economic losses by increasing birds.Polymerase chain reaction (PCR) (Saiki et mortality in chicken, diminished weight gain, al., 1985) has been applied as a rapid diagnostic poor feed conversion, drop in egg production and tool for the detection of avian viral and bacterial poor egg quality.It may also be involved in immunopathogens (Nguyen et al., 1994;Ganesh et al., suppression leading to increase incidence of 2002;Dahiya et al., 2002).secondary infection (Sambrook et al., 1989).This method is not only rapid, but also more A number of useful methods have been sensitive and specific than other diagnostic tests.developed for the diagnosis of avian adenovirus Utilizing most advanced techniques of molecular infections, including virus isolation in cell culture biology the hydroperi-cardium syndrome agent (Cowen et al., 1978), indirect immunofluocan be characterized in order to develop a highly rescent assay, virus neutralization (Adair et al., specific vaccine and in turn providing a quick, 1980;Adair et al., 1986), enzyme linked reliable and specific diagnosis.This study deals immunosorbant assay and the double immunewith the molecular characterization of avian diffusion (Adair et al., 1980;Adair et al., 1986; adenoviruses isolated in Anand Distrtict of Cowen, 1987).However, most of them are laborious and time-consuming.The main Gujarat through a combination of PCR and DNA problem with any serological test for adenovirus sequencing.

Materials and Methods
PRISMTM 310 Genetic Analyzer by using forward and reverse primers were assembled for Polymerase Chain Reaction: Viral DNA was analysis by SeqScapeV2.5 software programme extracted from infected liver sample using DNeasy and a consensus sequence was obtained.Consensus Tissue Kit (QIAGEN) as per the manufacturer's sequence thus obtained was aligned with various protocol.For PCR 2µl of DNA was amplified published sequences of the Hexon protein gene using 15pmol of each primer (Forward, in Genbank using NCBI Blast and RTT CAG RCA GAC GGT -3' nucleotide CLUSTAL W (1.82) software programmes.All positions 144-161; Reverse, 5'-TAG TGA TGM the nucleotide sequences were aligned for CGS GAC ATC AT -3' nucleotide positions 1041phylogenic analysis using the Clustal W program. 1021).The gene encoding the Hexon protein of fowl adenovirus group-I was chosen for the Results and Discussion selection of primers (Meulemans et al., 2001).
Detection of Fowl adenovirus by PCR: The PCR The amplification was carried out in thermocycler products generated was confirmed for their by initial denaturation at 94°C for 5min and 35 expected size (897 bp) in 2 % (w/v) agarose gel in cycles of 94°C for 2min, 60°C for 1 min and 72°C 0.5X TBE buffer as per the method of Sambrook for 90s, followed by final elongation at 72°C for et al. ( 1989) using horizontal submarine 2min.The amplified product was electrophoelectrophoresis apparatus (Bangalore Genei, retically separated in a submerged two percent India).The amplified PCR analyzed by Agarose agarose gel and visualized under ultraviolet light.
gel electrophoresis indicated a DNA fragment of DNA sequencing of field isolate: Primers and approximately 890 bp as expected by primer unincorporated dNTPs present in PCR product of HexonA F & HexonB R (Fig. 1).This was two field sample were removed by PCR compared with 500 bp ladder marker.No DNA purification kit (Perfectprep PCR cleanup 96-cat fragments were detected visually in ethidium no.955156013-Eppendorf). Cycle sequencing bromide stained agarose gel electrophoresis was performed following the instructions when PCR was carried out from DNA extracted supplied along with Big Dye® Terminator v3.1 from tissues of healthy bird.Cycle Sequencing Kit (Applied Biosystems).The reaction was carried out in a final reaction volume of 20 µl using 200 µl capacity thin wall PCR tube.The reaction was carried out in a final reaction volume of 20 µl using 200 µl capacity thin wall PCR tube.The tubes containing the mixture were tapped gently, spun briefly and then were transferred to thermal cycler.The cycling protocol was designed for 28 cycles with the thermal ramp rate of 100C per second as as Initial denaturation was carried out at 94°C for 5 min followed by 30 sec denaturation at 94°C, 10 sec at 60°C for annealing and 4 min at 72°C for extension.After the reaction, the extension products were purified sequence was done with foreign isolates and tree diagram created (Fig. 2).

DNA sequencing and phylogenetic analysis of
On phylogenetic analysis using Clustal W field isolate: Sequencing can be performed on program showing 3 major group like upper, any part of the genome, but was usually middle and lower respectively (Fig. 2).Within the performed on a selected part of the Hexon protein uppers group, 2 minor branches were observed.gene (Meulemans et al., 2001)

Fig 1 .
Fig 1. Agarose gel electrophoresis showing PCR amplified by using Ethanol-EDTA purification method.product (890) of field isolates.Electrophoresis and data analysis was carried out The similar result was obtained by on the automated ABI PRISMTM 310 Genetic Meulemans et al., (2001) using same primer.Analyzer (Applied Biosystems, USA) using PCR were record by Steer et al.,(2009) using appropriate Module, Basecaller, Dyeset/Primer same primer of present study of the 12 reference and Matrix files.The nucleotide sequences of the serotypes with the exception of FAdV-5, which Hexon protien gene obtained through ABI that of the TAdV3 sequence and M158-04 was Veterinary College, Anand Agricultural University, only 14.1%.With 12.5%, the sequence of EDSV Anand, Gujarat, for their help and support.showed the lowest percentage of identity when Conflict of interest compared with that of M158-04.A phylogenetic Authors declare that they have no conflict of tree was established, showed 3 major branches interest.for the proposed genera, Aviadenovirus (FAdV1-12), Atadenovirus (EDSV) and Siadenovirus.Within References the Aviadenovirus branch, 7 minor branches were 1. Adair, BM; McFerran, JB and Calvert, VM observed.However, within this major branch a (1980).Development of a microtitre sole minor branch was formed by M158-04 fluorescent antibody test for serological indicating that M158-04 represents a new avian detection of adenovirus infection in birds.adenovirus genotype.Avian Pathol., 9: 291-300.

Figure- 2
Figure-2 Phylogenetic tree based on nucleotide sequnces of Hexon Gene of field isolates and sequnces from Gene bank.