Download isolation of fowl adenovirus in chicken embryo liver cell culture and

Ind. J. Sci. Res. and Tech. 2014 2(3):33-36/Soumyalekshmi et al
Online Available at: http://www.indjsrt.com
Research Article
ISSN:-2321-9262 (Online)
ISOLATION OF FOWL ADENOVIRUS IN CHICKEN EMBRYO LIVER CELL
CULTURE AND ITS DETECTION BY HEXON GENE BASED PCR
*
Soumyalekshmi S.1, M. K. Ajith2, Meshram Chandraprakash3
Department of Animal Biotechnology, NDRI, Karnal, Haryana, India
2
Animal Nutrition Division, IVRI regional station, Palampur
3
Department of Animal Biotechnology, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar,
Haryana, India
*Author for Correspondence
ABSTRACT
Inclusion body hepatitis is a sporadic disease condition caused by several serotypes of fowl adenoviruses. In the
present study, Indian isolates of fowl adenovirus were propagated in primary chick embryo liver cell culture and the
virus was detected by hexon gene based polymerase chain reaction. 10-13 day old chicken embryos were dissected
to remove the liver and the liver tissue was subsequently trypsinised and processed for the preparation of chicken
embryo liver cell culture. Filtered liver homogenates from dead chickens suspected for avian adenovirus infection
was used as the virus inoculum for infecting the chicken embryo liver cell monolayers. The virus was subsequently
passaged and the cytopathic effects were detected from the 2nd passage onwards. The cytopathic effects observed
were in the form of rounding, clumping and detachment of cells subsequently leading to detachment of the entire
monolayer at 96 hrs of infection. The growth of the virus was detected by polymerase chain reaction based on hexon
gene and the cell culture supernatant from seventh passage of each isolate gave the specific PCR product proving the
ability of chicken embryo liver cell culture to be used as a suitable medium for the isolation and propagation of fowl
adenoviruses.
1
Key Words: Inclusion Body Hepatitis, Fowl Adenovirus, Primary Chicken Embryo Liver Cell Culture, Hexon Gene
and Polymerase Chain Reaction
INTRODUCTION
Fowl adenovirus (FAV) is a member of the family Adenoviridae, infecting chickens and responsible for a sporadic
disease condition called Inclusion Body Hepatitis (IBH). In India, IBH was first reported in 3-weeks old broiler
chicks, characterized by enlarged, mottled and friable liver with intranuclear inclusion bodies in the hepatocytes,
causing about 15% mortality (Grewal et al., 1981). IBH along with hydropericardium syndrome (IBH-HPS),
popularly called ‘litchi heart’ disease, has been reported to be particularly important in some countries in Asia and
America (Shane, 1996).
Isolation of the virus is an important and essential procedure for the subsequent identification and typing of the
virus. The fowl adenovirus can be grown in primary cell cultures of chicken kidney (Khaweja et al., 1988) or
chicken embryonic liver (CEL) cells (Naeem et al., 1995). CEL culture was used for the isolation of FAVs taken
from liver tissues of birds suffering from IBH-HPS from different farms of India (Kataria et al., 1996). Embryonated
eggs have also been reported as a sensitive medium for the isolation and propagation of FAVs, by inoculating liver
homogenate of HPS affected birds in 10–12 day old specific pathogen free (SPF) chicken embryos through
chorioallantoic sac route (Cowen and Braune, 1988; Abe et al., 1998) and 5-7 day old SPF chicken embryo via yolk
sac route (Cowen and Braune, 1988; Mazaheri et al., 1998). In the present study, four Indian isolates of fowl
adenovirus were propagated in primary chick embryo liver cell culture and the virus was detected by hexon gene
based polymerase chain reaction.
MATERIALS AND METHOD
Samples for virus isolation
Liver samples were collected from dead chickens brought for post-mortem examination and suspected for avian
adenovirus infection, in the Department of Veterinary Public Health and Epidemiology, Chaudhary Charan Singh
Haryana Agricultural University, Hisar during the period 2009-2010. The tissues were collected aseptically in sterile
vials containing 50% phosphate buffered glycerol and the vials were stored at -20 °C until further processing.
Detection of FAV hexon gene
For detection of FAV genes, 10% homogenates of the chicken liver tissues were prepared in lysis buffer. DNA was
extracted from the homogenates using conventional phenol: chloroform: isoamyl alcohol (PCI) method as per the
procedure recommended by Sambrook and Russel (Sambrook and Russell, 2001). For PCR H1/H2 primers
published by Raue and Hess (Raue and Hess, 1998), was used which can detect DNA with 1,219 bases from whole
33
Ind. J. Sci. Res. and Tech. 2014 2(3):33-36/Soumyalekshmi et al
Online Available at: http://www.indjsrt.com
Research Article
ISSN:-2321-9262 (Online)
hexon gene of FAV. Nucleotide sequence of the forward primer (H1) was 5´TGGACATGGGGGCGACCTA3´ and
that of the reverse primer (H2) was 5´AAGGGATTGACGTTGTCCA3´. PCR was done with Taq DNA polymerase
(Fermentas) under conditions of initial denaturation at 94 ºC, 5 min followed by 35 cycles of 45s at 94 ºC, 1m at 60
o
C and 1min at 72 oC, and final extension at 72 oC, 10min. The PCR products were detected by gel electrophoresis
with 1% Ultra Pure agarose (Invitrogen, USA) and 1 Kbp DNA ladder (Fermentas). Four samples which proved
positive for avian adenovirus were used for the preparation of inoculum for propagation of virus in CEL culture.
Preparation of chicken embryonic liver cell culture
Primary chicken embryo liver (CEL) cell culture was prepared from 10-13 days old embryonated chicken eggs as
per the method of Adair et al., (1979) with minor modifications. Liver from the embryos were removed aseptically
and washed twice with Hanks Balanced Salt solution (HBSS) (pH 7.4). The liver tissue was minced and washed
gently with HBSS and trypsinized gently with 0.25% trypsin-EDTA solution at 37 °C for 30m. The trypsinised cells
were decanted and filtered through sterile gauze tied to a sterile beaker. To stop the activity of residual trypsin,
growth medium, DMEM (Sigma) containing 10 % foetal calf serum (FCS) (Sigma) was added to the filtrate at the
rate of 10 ml/100 ml. The filtrate containing cells was centrifuged at 1500 rpm for 20 minutes at 4 °C and the cell
pellet obtained was washed twice in the growth medium. The live cell concentration was adjusted to 5 × 10 6 cells/
ml of the medium and cell suspension in 10 ml and 5 ml volumes were seeded in 75 cm2 and 25 cm2 tissue culture
flasks respectively and incubated at 37 °C in BOD incubator until confluent monolayer formed.
Isolation of the virus in CEL culture
20% suspension (w/v), of liver homogenate in phosphate buffered saline (PBS), pH 7.4, containing antibiotics
(penicillin 1000 IU/ml and streptomycin 1000 µg/ml) was prepared and subjected to 3 cycles of freezing and
thawing and centrifuged at 1500 rpm for 20 minutes at 4 °C. The supernatant obtained was filtered through 0.22µm
membrane filter (Millipore, USA). The filtrate was stored in sterile vials at -20 °C and used as inoculum for infection
of the primary chicken embryo liver cell culture.
Flasks containing complete monolayers were washed twice with HBSS and 500µl and 1ml of the liver tissue
homogenates were inoculated in 25 cm2 and 75 cm2 tissue culture flasks respectively. The virus inoculum was
allowed to adsorb onto the cells at 37ºC for 60 min. Following adsorption, maintenance medium containing 2% FCS
was added to the culture and the infected flasks were incubated at 37ºC and the monolayer was observed for
cytopathic effect (CPE) daily for about three days. After CPEs developed prominently, the cells were frozen and
thawed 3 times and viral fluid was harvested by centrifugation at 2,000 rpm, 10 min and stored at –20 oC for further
use and propagated FAVs was confirmed by PCR for hexon gene.
Confirmation of virus propagation by hexon gene based PCR
DNA was extracted from the cell culture supernatant using conventional phenol: chloroform: isoamyl alcohol (PCI)
method as per the procedure recommended by Sambrook and Russel (2001) with minor modifications. To confirm
the presence of virus in the cell culture supernatant, the extracted products were amplified by polymerase chain
reaction using primers H1/H2. The PCR products were detected by gel electrophoresis with 1% Ultra Pure agarose
(Invitrogen, USA) and 1 Kbp DNA ladder (Fermentas).
RESULTS AND DISCUSSION
The FAV produced CPE in chick embryo liver cell culture from the 2 nd passage onwards. The cytopathic effects
observed were in the form of rounding, clumping and detachment of cells, where as no such changes were observed
in the uninfected CEL cell cultures. Swelling and rounding of the infected cells appeared within 48 hrs of infection
and by 72 hrs post infection, the cells started detaching from the monolayer. Complete detachment of the
monolayers observed at 96 hrs of infection. Subsequent passages gave CPE within 24 to 48 hrs and involved
majority of the cells in monolayer. The microphotograph of uninfected primary chicken embryo liver cell culture
depicting multiplying liver cells and monolayer of CEL cell culture showing CPE are given in Fig. 1 and 2
respectively. Cell culture supernatant from seventh passage of each isolate was subjected to DNA extraction and
PCR amplification using primer pair H1/H2 and a specific product of 1219 bp was detected by 1% agarose gel
electrophoresis (Fig. 3). This confirmed the growth of the virus in the cell culture.
Many workers have attempted isolation of fowl adenovirus in CEL cell culture and succeeded in the adaptation of
the virus (Oberoi et al., 1996; Kataria et al., 1997; Barua and Rai, 2003). In the present study, the FAV was
propagated in primary chick embryo liver cell culture and the virus produced rounding, degeneration and
detachment of the infected cells from the monolayer, similar to the observations of many earlier workers (Adair et
al., 1979; Oberoi et al., 1996; Kataria et al., 1997; Balamurugan et al., 2002). The cell culture as template fluid was
used to amplify 1219 bp FAV DNA product and this confirmed the virus in infected cell culture fluid.
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Ind. J. Sci. Res. and Tech. 2014 2(3):33-36/Soumyalekshmi et al
Online Available at: http://www.indjsrt.com
Research Article
Figure 1: Microphotograph of primary chicken
embryo liver cell culture depicting multiplying liver
cells in a monolayer after 48 hours of growth
ISSN:-2321-9262 (Online)
Figure 2: Microphotograph of primary chicken
embryo liver cell culture showing rounding,
clumping and detachment of cells after 72hrs of
infection
Figure 3: Detection of viral DNA in cell culture supernatant by hexon gene specific PCR assay with primer
pair H1/H2 amplifying an expected product of 1219bp. Lane M- 100bp DNA ladder (Fermentas); Lanes 1, 2, 3,
4- Samples; Lane 5- Positive control; Lane 6- Negative cell culture control
CONCLUSION
The better growth of FAV in CEL cell culture may be attributed to the fact that the predilection site of FAVs is the
nucleus of hepatocytes. The present study proves that the CEL cell culture is a very suitable medium for the
isolation and propagation of FAVs.
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