sequence analysis, pathogenicity and cytokine gene
TRANSCRIPT
Sequence analysis, pathogenicity and cytokine gene expression patterns
associated with fowl adenovirus infection
by
Helena Grgić
A Thesis presented to
The University of Guelph
In partial fulfillment of requirements for the degree of
Doctor of Philosophy in
Pathobiology
Guelph, Ontario, Canada © Helena Grgić, May, 2012
ABSTRACT
SEQUENCE ANALYSIS, PATHOGENICITY AND CYTOKINE GENE
EXPRESSION PATTERNS ASSOCIATED WITH FOWL ADENOVIRUS
INFECTION
Helena Grgić Advisor: University of Guelph, 2012 Dr. Éva Nagy
The family Adenoviridae consists of five genera, including the genus
Aviadenovirus, which infects avian species. The genus Aviadenovirus currently
comprises five fowl (Fowl adenovirus A-E), one falcon (Falcon adenovirus A), and one
goose (Goose adenovirus) adenovirus species. Fowl adenoviruses (FAdVs) have a
worldwide distribution. Some are associated with diseases such as inclusion body
hepatitis (IBH), while FAdV species C serotype 4 (FAdV-4) has been associated with
hydropericardium-hepatitis syndrome (HHS).
In this study, the complete nucleotide sequence of fowl adenovirus serotype 8
(FAdV-8) was determined. The full genome was 44,055 nucleotides (nt) in length, with
an organization similar to that of the FAdV-1 and FAdV-9 genomes. No regions
homologous to early regions E1, E3, and E4 of mastadenoviruses were recognized
Pathogenicity of FAdV-8 and FAdV-4 were studied in specific-pathogen-free
chickens following oral and intramuscular inoculations. Pathogenicity was determined on
the basis of clinical signs and gross and histological lesions. Additionally, virus shedding
and viral genome copy numbers in liver, cecal tonsil, and bursa of Fabricius were
determined.
The role of interleukins (IL) in the pathogenicity of and immune response to
FAdVs is unknown. Therefore, in a chicken experiment, interferon-γ, IL-10, IL-18, and
IL-8 gene expression was evaluated following FAdV-8 and FAdV-4 infection. Cytokine
gene expression was examined in the liver, spleen, and cecal tonsils. This study explored
the ability of fowl adenoviruses to subvert the host cell’s secretion of cytokines in
response to infection as an important viral mechanism for immune evasion during
infection.
Variations in virulence of FAdVs are likely to be determined by the fiber alone as
shown by Pallister et al. (1996). Therefore, we compared and analyzed the nt and amino
acid (aa) sequences of the fiber gene of pathogenic and non-pathogenic FAdVs
representing species groups D (FAdV-11) and E (FAdV-8). According to our data,
virulence might not be associated only with sequence of the fiber gene.
This work is a continuation of our efforts towards better understanding of the
molecular biology of FAdVs and the pathogenesis of the disease, with an emphasis on the
role of interleukins, an unknown area.
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ACKNOWLEDGEMENTS
I wish to express my sincere appreciation and thanks to my advisor Dr. Ѐva
Nagy, for her support and guidance through the past several years and for keeping on
pushing me whenever I hit a bump in my research. I am also very thankful to my
committee members Drs. Annette Nassuth and Sarah Wootton for the many thoughtful
comments, ideas and suggestions that have greatly improved this thesis.
Above all, I would like to acknowledge the invaluable support of Dr. Bruce
Hunter, my mentor and friend, who taught me everything I know about poultry pathology
and histology. Bruce was always approachable, always generous with his time, always
ready to help and encourage. I will miss him greatly for his knowledge and expertise, but
even more for his kind and gentle nature.
I thank to Dr. Shayan Sharif for his constructive criticism and comments on my
cytokine research and for the willingness to let me use his lab.
Many of the current and past Virology lab members have become dear friends
over the years and I will miss all of them, including the rare lab drama. Sheila, Rita, Juan,
Andres, Bryan, Rasha, Li, Betty-Anne, thank for brightening many of my days in lab.
Most of all I would like to thank my family, my husband Zvonimir and our
daughter Korina: I can’t thank you enough for your endless support and patience through
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this process and for being the best two distractions from work one could wish for. I could
not have done this without you.
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TABLE OF CONTENTS
ABSTRACT ....................................................................................................................... ii
ACKNOWLEDGEMENTS ............................................................................................ iv
LIST OF FIGURES ......................................................................................................... ix
LIST OF ABBREVIATIONS .......................................................................................... x
CHAPTER 1 ...................................................................................................................... 1
LITERATURE REVIEW ....................................................................................................... 1 1. Diseases caused by avian adenoviruses .................................................................. 1
1.1. Inclusion body hepatitis ................................................................................... 1 1.1.1. Disease ...................................................................................................... 1 1.1.2. Clinical signs and pathogenesis ................................................................ 1 1.1.3. Gross lesions and histopathology.............................................................. 2 1.1.4. Virus excretion and transmission .............................................................. 4
1.2. Hydropericardium-hepatitis syndrome ............................................................ 5 1.2.1. Disease ...................................................................................................... 5 1.2.2. Clinical signs ............................................................................................. 6 1.2.3. Gross lesion and histopathology ............................................................... 7 1.2.4. Virus excretion and transmission .............................................................. 8
1.3. Other diseases caused by avian adenoviruses .................................................. 8 1.4. Diseases in birds other than poultry ............................................................... 10 1.5. Diagnosis of aviadenovirus infection............................................................. 12 1.6. Control ........................................................................................................... 13
2. Adenoviruses ......................................................................................................... 14 2.1. Taxonomy ...................................................................................................... 14 2.2. Characteristics of adenoviruses ...................................................................... 15
2.2.1. Structure and physicochemical composition of the adenovirus .............. 15 2.2.2. Proteins ................................................................................................... 16 2.2.3. Genome ................................................................................................... 20
2.3. Adenovirus replication cycle ......................................................................... 24 2.3.1. Attachment and penetration .................................................................... 24 2.3.2. Activation of early viral genes ................................................................ 27 2.3.3. DNA replication ...................................................................................... 28 2.3.4. Late gene expression ............................................................................... 29 2.3.5. Assembly and release .............................................................................. 29
3. Avian immune response ........................................................................................ 30 3.1. Innate immune response ................................................................................ 32
3.1.1. Cytokines ................................................................................................ 37 3.2. Adaptive immunity ........................................................................................ 40
HYPOTHESIS AND OBJECTIVES ....................................................................................... 43
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CHAPTER 2. Pathogenicity and complete genome sequence of a fowl adenovirus serotype 8 isolate ............................................................................................................. 46
Abstract ............................................................................................................. 47 1. Introduction ............................................................................................... 49 2. Materials and methods .............................................................................. 51 3. Results ....................................................................................................... 54 4. Discussion ................................................................................................. 59
CHAPTER 3. Key cytokines activated during infection of chickens with a serotype 8 fowl adenovirus ............................................................................................................... 71
Abstract ............................................................................................................. 72 1. Introduction .............................................................................................. 74 2. Materials and methods ............................................................................. 76 3. Results ...................................................................................................... 79 4. Discussion ................................................................................................ 81
CHAPTER 4. Pathogenicity and cytokine gene expression patterns of a serotype 4 fowl adenovirus isolate, a vaccine candidate ................................................................ 97
Abstract ............................................................................................................. 98 1. Introduction ............................................................................................ 100 2. Material and methods ............................................................................. 101 3. Results .................................................................................................... 106 4. Discussion .............................................................................................. 109
CHAPTER 5. Molecular characterization of the fiber gene of fowl adenovirus serotypes 8 and 11 ......................................................................................................... 123
Abstract ........................................................................................................... 124
GENERAL DISCUSSION ........................................................................................... 141
References ...................................................................................................................... 148
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LIST OF TABLES Table 2. 1. Percent identities/similarities of selected FAdV-8 proteins and their
homologues ............................................................................................................... 65 Table 2. 2. Viral genome copy numbers in tissues of chickens inoculated with FAdV-8
................................................................................................................................... 66 Table 2. 3. Virus titers in the feces of chickens inoculated with FAdV-8 ....................... 67 Supplementary Table 2. 1. Position of 46 predicted genes of the FAdV-8 genome ...... 68 Table 4. 1. Viral genome copy numbers in tissues of chickens inoculated with FAdV-4
................................................................................................................................. 115 Table 4. 2. Virus titers in the feces of chickens inoculated with FAdV-4 ..................... 116 Table 5. 1. Description of the isolates. ........................................................................... 133 Table 5. 2. Percent identities/similarities between FAdV-8 and FAdV-11 fiber proteins
and their homologues. ............................................................................................. 134 Table 5. 3. Differences in amino acids between FAdV-8 isolates. ................................ 135
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LIST OF FIGURES
Figure 1. 1. Schematic transection of the adenovirus particle ......................................... 23 Figure 1. 2. Reproductive cycle of human adenovirus type 2. ........................................ 26 Figure 2. 1. Schematic representation of FAdV-8, FAdV-9 and FAdV-1 genomes,
members of the genus Aviadenovirus ....................................................................... 69 Figure 2. 2. Antibody response to viral proteins in chickens inoculated with FAdV-8 by
the oral and intramuscular routes and mock infected as measured by S/P ratios ..... 70
Figure 3. 1. Cytokine mRNA expression in CH-SAH following FAdV-8 infection ....... 89 Figure 3. 2. Cytokine mRNA expression in spleen of chickens infected with FAdV-8. . 91 Figure 3. 3. Cytokine mRNA expression in cecal tonsil of chickens infected with FAdV-
8................................................................................................................................. 93 Figure 3. 4. Cytokine mRNA expression in liver of chickens infected with FAdV-8 ..... 95 Figure 4. 1. Antibody response to viral proteins in chickens inoculated with FAdV-4 by
the oral and intramuscular routes and mock infected as measured by S/P ratios. Panel A. Serum samples were tested against homologous virus, FAdV-4. Panel B. Serum samples were tested against heterologous virus, FAdV-9 ........................... 117
Figure 4. 2. Cytokine mRNA expression in cecal tonsil of chickens infected with FAdV-4............................................................................................................................... 119
Figure 4. 3. Cytokine mRNA expression in liver of chickens infected with FAdV-4 ... 120 Figure 4. 4. Cytokine mRNA expression in spleen of chickens infected with FAdV-4..
................................................................................................................................. 122 Figure 5. 1. Amino acid sequence alignment of the tail sequence of FAdV-8 and FAdV-
11 isolates made by CLUSTALW .......................................................................... 136 Figure 5. 2. Regions of FAdV-8 and FAdV-11 fibers ................................................... 137 Figure 5. 3. Supplementary data. Amino acid alignment of FAdV-8 fibers derived from
flocks with and without signs of IBH ..................................................................... 139 Figure 5. 4. Supplementary data. Amino acid alignment of FAdV-11 fibers derived from
flocks with and without signs of IBH ..................................................................... 140
x
LIST OF ABBREVIATIONS vßs Alphavbeta integrins AAdV Avian adenoviruses AASV Avian adenovirus splenomegaly virus AAV Adeno-associated virus Ab Antibody ADP Adenovirus death protein AGID Agar gel immunodiffusion APC Antigen presenting cells CAdV Canine adenovirus CAR Coxsackie and adenovirus receptor CEK Chicken embryo kidney CEL Chicken embryo liver CELO Chicken embryo lethal orphan CIAV Chicken infectious anemia virus CK Chicken kidney CMV Cytomegalovirus CPE Cytopathic effect CSIF Cytokine-synthesis inhibitory factor DAdV Duck adenovirus DBP DNA-binding protein DC Dendritic cells DNA Deoxyribonucleic acid ds double-stranded d.p.i. days post infection EDS Egg drop syndrome EDSV Egg drop syndrome virus EID Embryonic development ELISA Enzyme linked immunosorbent assay EM Electron microscopy ER Endoplasmic reticulum FaAdV Falcon adenovirus FAdV Fowl adenovirus GALT Gut associated lymphoid tissue GoAdV Goose adenovirus HAdV Human adenovirus HEV Hemorrhagic enteritis virus HHS Hydropericardium-hepatitis syndrome HIV-1 Human immunodeficiency virus 1 h.p.i. hour post infection HPS Hydropericardium syndrome HPV Human papilloma virus hr hour HSP Heat-shock protein
xi
IBDV Infectious bursal disease virus IBH Inclusion body hepatitis IBV Infectious bronchitis virus Ig Immunoglobulin IFN Interferon ITRs Inverted terminal repeats LRR Leucine rich repeat motif MALT Mucosal associated lymphoid tissue MDV Marek’s disease virus ml Milliliter MHC Major histocompatibility complex MMTV Mouse mammary tumor virus MSB-1 Marek’s disease lymphoblastoid cell NF-1 Nuclear factor I NK Natural killer NLRP Nod-like receptor NOD Nucleotide-binding oligomerization domain nt Nucleotide NTR N-terminal region Oct-1 Octamer-binding protein ORF Open reading frame PAMPs Pathogen associated molecular patterns PiAdV Pigeon adenovirus Pol Polymerase PRRs Pattern recognition receptors pTP Pre-terminal protein RE Restriction enzyme RGD Arg-Gly-Asp motif RFLP Restriction fragment length polymorphism RNA Ribonucleic acide ROS Ractive oxygen species RSV Respiratory syncytial virus RT Reverse transcription SDS Sodium dodecyl sulfate SPF Specific pathogen free ss single-stranded TAdV Turkey adenovirus TCID50 50% tissue culture infective dose TLR Toll-like receptors TP Terminal protein TR Tandem repeat wt Wild type
1
CHAPTER 1
Literature Review 1. Diseases caused by avian adenoviruses 1.1. Inclusion body hepatitis 1.1.1. Disease
Inclusion body hepatitis (IBH) was first described by Helmboldt and Frazier
(1963). The syndrome is characterized by sudden death and massive liver necrosis and
the disease is mainly diagnosed in broiler chickens. Birds are usually found dead, but
occasionally are seen in an extremely depressed condition shortly before death. Death
occurs within a few hours after initial signs. Outbreaks are usually observed at two to
three weeks of age and last up to three weeks, with total losses as high as 8%. In the last
two decades, IBH has been reported in broiler flocks in almost every country around the
world (Philippe et al., 2005; Gomis et al., 2006; Goodwin et al., 1993; Young et al.,
1972; Saiffudin and Wilks, 1991; Erny et al., 1991). Although adenoviruses are
associated with numerous clinical conditions, their primary role in disease is still not
understood. Most outbreaks are described in broiler flocks, but outbreaks in pullets and
replacement layers, respectively are reported by Bickford (1974) and Hoffmann et al.
(1975).
1.1.2. Clinical signs and pathogenesis
Mortality is closely related to the pathogenicity of the virus, secondary infection with
other pathogens, and susceptibility of the chickens. In Canada, the most commonly
isolated serotypes are 2, 7, 8, and 11 (Ojkic et al., 2008b). From the available evidence, it
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seems that most of the strains of fowl adenoviruses (FAdVs) follow the same pattern of
infection. Following initial multiplication, viraemia occurs and results in virus spread to
virtually all organs. Viraemia is detected from 24 hours post inoculation (h.p.i.), with two
peaks, one at day 2 and the second at day 7 post infection (p.i.) (Saifuddin and Wilks,
1991). The virus has been detected in pancreas, bursa of Fabricius, esophagus, trachea,
spleen, proventriculus, bone marrow, thymus, and kidney. The main sites of virus
replication are the respiratory and alimentary systems, including the caeca (Saiffudin and
Wilks, 1991). In chickens, immunosuppression caused by other pathogens can influence
the clinical severity of adenoviral infection (Adair and Fitzgerald, 2008). It is apparent
that impairment of the bursa of Fabricius by infectious bursal disease virus (IBDV)
facilitates adenoviruses in producing IBH (Fadly et al., 1975). The ability of IBDV to
enhance the pathogenesis of IBH leads to the conclusion that field cases of IBH must be
preceded by a stress on the humoral immune response (Fadly et al., 1975). Another
commonly known immunosuppressant is chicken infectious anemia virus (CIAV), proven
to be a potent immunosuppressive agent for very young unprotected chicks and therefore
increasing their susceptibility to secondary infections in general (Adair and Fitzgerald,
2008). Beside viruses, stress is one of the most common causes of immunosuppression in
poultry. It is believed that the negative economic impact of IBH is usually enhanced in
stressed birds (Adair and Fitzgerald, 2008).
1.1.3. Gross lesions and histopathology
At gross examination, there is marked anemia, icterus of the skin and
subcutaneous fat deposits, haemorrhages in various organs, and pale inactive bone
3
marrow. The main lesion is in the liver, which is swollen and light brown to yellow,
occasionally with haemorrhages (Adair and Fitzgerald, 2008).
At histopathological examination, large, round, eosinophilic inclusion bodies with
clear halos are present in hepatocytes (Grimes et al., 1977a; Itakura et al., 1974).
However, some researchers reported that basophilic inclusions may occasionally be seen
(Grimes et al., 1977b; McCracken et al., 1976). Small focal haemorrhages and nephrosis
are reported in the kidney (Howell et al., 1970). Moreover, the first signs of infection can
be seen in hepatocytes between 16 and 20 h.p.i. and consist of a ring or a partial ring of
medium electron density (Sharpless and Jungherr, 1961). Similar structures have been
observed in cells infected with human adenovirus (HAdV) type 12 (Martinez-Palomo et
al., 1967). Early morphological lesions of infected hepatocytes are observed in the
nucleus (nuclear and nucleolar hypertrophy) 8 hr after virus inoculation (Martinez-
Palomo et al., 1967). At 18 h.p.i., irregular patches of dense material in the nucleoplasm
are reported, differentiated into type I and II inclusions depending on their structure.
Observed inclusions increase rapidly in size and virus particles start to appear between 20
and 30 h.p.i. In general, viral particles tend to be located in the vicinity of type II
inclusions. In the following step, the nuclei of infected cells show large round foci (type
III inclusions), which are larger than the other two types of inclusions. Type III inclusions
gradually fill up most of the nucleus. At 30 h.p.i., the proportions of all three types of
inclusions increase and viral particles develop into crystalline formations (Martinez-
Palomo et al., 1967). By 36 h.p.i., viral crystalline formations are commonly seen and a
dense reticulated substance appears, designated as type IV inclusions. Disorganization of
the normal architecture of the nucleus in most cells is observed 40 h.p.i.. During the final
4
stage of infection, the nucleus is enlarged, the nuclear membrane is distorted, and virus
particles are arranged in crystalline formations which increase in size until disruption of
the nucleus. Lysis of infected cells occurs at the 3rd day after viral inoculation (Martinez-
Palomo et al., 1967).
Cytopathological examination of the gastrointestinal tracts of 1-day-old chicks
infected with avian adenovirus strain 93 shows that antigen is present only in the
epithelial cells on the surface of the villi. Approximately 50% of the infected cells are
detected in the proximal third of the villus, while 25% occured in the middle and another
25% in the distal third (Clemmer and Ichinose, 1968). Both columnar and goblet cells are
infected. The most prominent change is hypertrophy of the epithelial cells with
enlargement of the nuclei. The nuclei are oval with margination of chromatin along the
interior surface of the nuclear membrane (Clemmer and Ichinose, 1968).
1.1.4. Virus excretion and transmission
FAdVs can be transmitted horizontally and vertically. Horizontal spread of the
virus occurs mainly via the feces, tracheal and nasal mucosa, and semen. FAdVs are most
commonly transmitted by the oral-fecal route (Adair and Fitzgerald, 2008; Grgic et al.,
2006).Various studies clearly showed that excretion in the feces, and to a lesser extent in
naso-oral secretions, are the major routes for virus spread. Clemmer and Ichinose (1968)
recorded peak fecal titers of 104.7 50% tissue culture infective doses (TCID50) per g at 4
d.p.i. and excretion continued up to 14 d.p.i. Virus isolation from layer replacements is
most successful when birds are 8 to 14 weeks old and then decreases with increasing age
5
until 34 weeks when virus can no longer be isolated (Cowen et al., 1977). FAdVs are
isolated from asymptomatic chickens as well, and the majority of these viruses induce
cytopathic effect (CPE) by the second passage in chicken kidney cell culture (Cowen et
al., 1977). Also, it is not uncommon to isolate multiple serotypes from one bird,
suggesting that there is little cross protection (Cowen et al., 1977). Therefore, it is not
unexpected to have birds that excrete one serotype in spite of high levels of neutralizing
antibody to another serotype (Cowen et al., 1977). The superiority of cloacal swabs over
tracheal swabs for adenovirus isolation is confirmed by Cowen et al. (1977). Vertical
transmission is considered an important factor in the epidemiology of IBH (McFerran and
Smyth, 2000). Although contradictory reports have been published over the years about
the role and model of vertical transmission of FAdVs (Cowen et al., 1978; Reece et al.,
1985), a recent publication confirmed vertical transmission and establishment of latent
infection with FAdVs (Grgic et al., 2006).
1.2. Hydropericardium-hepatitis syndrome 1.2.1. Disease
Fowl adenovirus C serotype 4 plays a major role in the aetiology of a broiler
disease called infectious hydropericardium-hepatitis syndrome (HHS), an emerging
disease of broiler chickens initially reported from Angara Goth near Karachi, Pakistan
(Khawaja et al., 1988). In addition to India and Pakistan, outbreaks have now also been
reported in the Middle East, Japan, Mexico, Peru, Ecuador and Chile (Abe et al., 1998;
Hess et al., 1999; Toro et al., 1999).
Initially it was hypothesized that HHS is caused by toxicity or by a nutritional
disorder. Investigated causative factors were mycotoxins, phycotoxins, and chemicals
6
such as chloride, polychlorinated biphenyls, and chlordane, which have all been
associated with hydropericardium (Jaffery, 1988). However, all attempts to reproduce the
disease with feed samples from infected farms failed. Therefore, an infectious agent was
suggested as the probable cause (Abdul-Aziz and Al-Attar, 1991). Based on successful
isolation of an agent from the liver, heart, and kidneys, and on transmission of disease
after subcutaneous inoculation of infected liver homogenate, the aetiological agent was
tentatively designated as an adenovirus (Khawaja et al., 1988). Subsequent studies
demonstrated typical icosahedral adenovirus particles in the purified liver extracts by
negative-staining electron microscopy. Additionally, basophilic intranuclear inclusion
bodies in the hepatocytes supported the view that the disease is caused by adenovirus
(Mazaheri et al., 1998).
1.2.2. Clinical signs
The disease is prevalent in 3- to 7-week-old broiler chickens. In field outbreaks,
the affected birds may not exhibit signs other than a high mortality, up to 75% (Jaffery,
1988). However, in the terminal stage of the disease, some birds might become dull,
depressed, huddle in corners with ruffled feathers, and exhibit a characteristic posture,
with the chest and beak resting on the ground (Anjum, 1990). The disease is observed in
healthy broilers showing a good growth rate, suggesting that rapid growth rate may be a
factor that predisposes broilers to HHS (Shafique and Shakoori, 1994). Rare outbreaks
are also recorded in broiler breeder flocks (32 weeks old) and commercial layers (17
weeks old), with up to 8% mortality (Anjum, 1990). Many researchers have reported
equal susceptibility of different broiler strains in both field and in experimental cases.
7
The Hubbard strain of broilers is reported as the most susceptible, followed by Indian
River and Lohmann strains (Khan et al., 1995).
1.2.3. Gross lesion and histopathology
The predominant gross lesion is hydropericardium, characterized by the
accumulation of a jelly-like fluid in the pericardial sac. The heart appears misshapen and
flabby. The pericardial sac has a balloon-like appearance and may contain up to 20 ml of
clear straw-colored fluid (Kumar et al., 1997). Other changes are a pale, swollen, and
friable liver with multifocal necrosis, congested lungs, and pale, swollen kidneys
containing urates in the tubules and ureters, and yellowish discoloration and petechial
haemorrhages in pericardial fat (Kumar et al., 1997; Nakamura et al., 1999).
At histopathological examination, the most consistent findings in the liver are
small multifocal areas of necrosis and mononuclear cell infiltration, with basophilic
intranuclear inclusion bodies in hepatocytes surrounded by a clear halo or filling the
entire nucleus (Nakamura et al., 1999). Microscopic changes in the heart are
mononuclear cell infiltration, severe vascular changes and disruption and separation of
muscle bundles and fibers due to oedema (Kumar et al., 1997). The most prominent
changes in the lung are congestion, oedema, and mononuclear cell infiltration with severe
vascular changes and haemorrhagic exudates in the bronchi and alveoli (Kumar et al.,
1997). In the kidneys, massive swelling of the tubular epithelium, necrosis, and extensive
haemorrhages are reported (Kumar et al., 1997). Changes in the bursa of Fabricius,
thymus, and spleen are lymphocytolysis and cyst formation that consequently lead to
8
lymphocyte depletion in the medullae of the follicles in the bursa of Fabricius. Marked
congestion and mild lymphoid cell depletion are characteristic changes in the thymus
(Kumar et al., 1997; Abdul-Aziz and Hasan, 1995). The only differences between HHS
and IBH are mortality rate and the presence of hydropericardium, which is the most
characteristic gross change in HHS. Hydropericardium alone depresses cardiac function
and produces muffling of the heart sounds (Nakamura et al., 2002).
1.2.4. Virus excretion and transmission
The HHS virus is transmitted vertically and horizontally among birds by the oral-
fecal route (Toro et al., 2001; Cowen, 1992)
1.3. Other diseases caused by avian adenoviruses
There are several other important diseases affecting poultry which are caused by
viruses belonging to the genera Siadenovirus and Atadenovirus of the family
Adenoviridae. Viruses in the genus Siadenovirus typically produce disease without
immunosuppressive factors, virus from the genus Atadenovirus cause egg drop syndrome
(Adair and Fitzgerald, 2008).
Hemorrhagic enteritis virus
Turkey adenovirus 3 (TAdV-3), also known as hemorrhagic enteritis virus (HEV)
of turkeys, belongs to the genus Siadenovirus. Virus is transmitted by the oral-fecal route
and infects turkeys at 4 weeks and older. The clinical signs of infection include
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depression, bloody droppings, and death. Usually the disease persists in an affected flock
7 to 10 days (Itakura and Carlson, 1975).
Splenomegaly in chickens
Avian adenovirus splenomegaly virus (AASV) of chickens belongs to the genus
Siadenovirus and initially was reported in 1979 in the southeastern U.S.A. Splenomegaly
in chickens is a subclinical disease, although pulmonary oedema, congestion, depression,
weakness, and progressive dyspnea are reported by Fitzgerald and Reed (1989).
Egg drop syndrome
Egg drop syndrome virus (EDSV) infects chickens, quail, geese, and ducks, and
belongs to the genus Atadenovirus (Adair and Fitzgerald, 2008). This virus causes high
losses in egg production. One of the first signs of infection is loss of color in pigmented
eggs and this is quickly followed by production of thin-shelled, soft-shelled, or shell-less
eggs. EDSV is transmitted vertically and horizontally through the drinking water
contaminated by fecal material (Cook and Darbyshire, 1980).
Gizzard erosion
Natural outbreaks of gizzard erosions in broilers are characterized by mortality in
young broilers without clinical signs of infection (Manarolla et al., 2009). On gross
examination, distended gizzards with haemorrhagic fluid and multiple black patchy
erosions are found (Abe et al., 2001). Some researchers also reported pancreatitis,
hepatitis, cholecystitis, and cholangitis (Ono et al., 2004).
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1.4. Diseases in birds other than poultry
Over the past few decades, adenoviruses have been described and isolated from a
wide variety of other avian species, such as waterfowl, game birds, cage birds, and
raptors (Adair and Fitzgerald, 2008).
Pigeon adenoviruses are associated with two distinct diseases, both causing
serious harm to the racing pigeon population. The first disease, called adenovirosis type I
or ‘classical adenovirosis,’ occurs when young pigeons first participate in races at the age
of 6 months. Pigeons are infected orally by cross-contamination in common baskets.
Consequently, virus is shed in feces, thereby rapidly infecting all other susceptible
pigeons in the loft. Only birds less than 1 year of age develop clinical signs, while older
pigeons remain clinically healthy. The virus replicates in the liver and in the nucleus of
intestinal epithelial cells, causing severe intestinal damage with extensive loss of proteins
and ions. The enhanced permeability of the intestinal wall, with bacterial overgrowth in
the intestinal lumen, creates a serious risk of septicaemia. Adenovirosis type II or
‘necrotizing hepatitis’ is characterized by sudden death in pigeons of all ages throughout
the year (De Herdt et al., 1995). Clinical signs are minimal, since birds die within 24 to
48 hr. Occasional clinical signs are vomiting and production of yellow, liquid droppings.
Mortality is approximately 30%, but in some cases can reach 100%. Histological
examination consistently reveals the presence of nuclear inclusion bodies in hepatocytes
and extensive hepatic necrosis. All conditions related to pigeon adenovirus infection are
reviewed by Vereecken et al. (1998). Growth analysis of adenoviruses isolated from
pigeons in chicken cells is described by Hess et al. (1998).
11
Adenovirus is also implicated in guineafowl disease. The virus was isolated from
the pancreas of 2-week-old guinea fowl affected with acute necrotizing pancreatitis with
prominent viral inclusions by Zellen et al. (1989). Gross examination showed emaciation
of all affected guinea fowl, with an enlarged, hard, and nodular pancreas, resulting in
distortion of the duodenal loop. Microscopic findings are generalized pancreatic necrosis
with large basophilic intranuclear inclusion bodies.
With the rapid expansion of the ostrich farming industry there is a consequent
increase in disease problems similar to those that are observed in the poultry industry.
Isolation of six adenoviruses from ostriches and their identification using restriction
endonuclease (RE) analysis is reported by Gough et al. (1997). RE analysis reveals that
five of the isolated viruses have a close genetic relationship to the FAdV-8 serotype (TR
59), while one isolate recovered from the pancreas of a 6-week-old ostrich with gross
lesions of pancreatitis has an RE pattern similar to the FAdV-2 serotype. In Italy, Capua
et al. (1994) described pancreatitis-like lesions in an ostrich chick from which an
adenovirus was isolated. Moreover, adenoviruses from ostriches can be transmitted to
guinea fowl, resulting in deaths and associated pancreatitis (Capua et al., 1994).
Falcon adenovirus (FaAdV-1) is a new species in the genus Aviadenovirus with
close similarity to FAdV-1 and FAdV-4 (for more detail see sections 2.1. and 2.2.2.). The
penton base of the FaAdV-1 lacks the Arg-Gly-Asp (RGD) and Leu-Asp-Val (LDV)
recognition motifs for integrin binding, suggesting that cell entry depends on a different
receptor. FaAdV-1 has broad cell tropism and is a primary pathogen that can infect
12
several species of falcons. The virus is associated with outbreaks of disease in captive-
raised nestling Northern Aplomado falcons, peregrine falcons, a Vanuatu falcon, Taita
falcons, orange-breasted falcons, and a merlin (Schrenzel et al., 2005; Van Wettere et al.,
2005). The primary lesions are inclusion body hepatitis, splenomegaly, and moderate to
severe lymphoid atrophy and lympholysis in the bursa of Fabricius. Virus is spread
horizontally (Schrenzel et al., 2005). The peregrine falcon is the primary reservoir host
for the virus, and FaAdV-1 replicates in cell cultures of falcon origin (Oaks et al., 2005).
The only other avian species that is seropositive to FaAdV-1 is the single barred owl.
Interestingly, falcons and owls are phylogenetically disparate, but share susceptibility to
another typically host-specific virus, columbid herpesvirus 1 (Sileo et al., 1983; Aini et
al., 1993).
1.5. Diagnosis of aviadenovirus infection
Laboratory diagnosis is based on direct detection, including isolation and
identification of the causative agent, or indirect detection with antibodies (Abs). Usually
the diagnosis of adenovirus infection in poultry is based on histology and detection of
intranuclear inclusion bodies in hepatocytes, or on detection of the antigen or virus
particles using an immunofluorescence test or electron microscopy. During the last two
decades, several molecular biological tools, such as PCR, real-time PCR, and RE analysis
have been developed for detection of viral DNA (Erny et al., 1991; Raue and Hess, 1998;
Jiang et al., 1999; Hess et al., 1999; Hess, 2000; Raue et al., 2002; Luschow et al., 2007;
Steer et al., 2009; Romanova et al., 2009). Most avian adenovirus PCRs utilizes the
hexon gene for primer design. The hexon consists of conserved regions (pedestals P1 and
13
P2) located inside the virion and variable loops (L1 to L4) that protrude from the surface
and contain type-specific neutralizing epitopes (Toogood et al., 1992). Consequently,
hexon pedestal and loop sequences permit the design of oligonucleotides that generate a
PCR product from all twelve serotypes; an RE analysis of the amplified PCR product
may be conducted to differentiate the serotypes. However, virus isolation from chicken
kidney (CK) or chicken embryo liver (CEL) cells is an essential procedure for further
typing of FAdVs (Sharpless et al., 1961). Embryonated eggs injected via the yolk sac
route are also a sensitive medium (Cowen, 1988). A group-specific ELISA is suitable to
detect less than 100 mean tissue-culture infective doses of adenovirus per gram of liver
tissue (Saiffudin and Wilks, 1991). The most common serologic test is immunodiffusion,
but its sensitivity is lower than the ELISA’s.
1.6. Control
Biosecurity practices are the most essential step to prevent infection. All factors
related to proper management including cleaning and disinfection of premises and
equipment, restricted entry of visitors and vaccination crews in the poultry houses play a
considerable role in prevention of the disease (Adair and Fitzgerald, 2008).
Immunosuppressive viruses such as IBDV and CIAV act as predisposing factors for
FAdV outbreaks (Adair and Fitzgerald, 2008). Therefore, control or elimination of
immunosuppressive pathogens is another important step in management procedures.
Despite the relatively high mortality and economic losses in the past several years in the
Canadian broiler industry due to IBH, no commercial vaccines are available in Canada,
but they have been successfully used in other countries. Comparison of two inactivated
14
vaccines prepared from the livers of infected chickens, one without adjuvant and another
with a liquid paraffin adjuvant, showed that oil emulsion vaccine is highly effective (Roy
et al., 1999). Additionally, inactivated chicken liver cell culture and embryonated egg-
propagated vaccine for subcutaneous application also provided sufficient protection
against infection (Naeem et al., 1995). Evaluation of five inactivated vaccines used in
Mexico confirmed complete protection with the absence of histological changes in organs
of challenged chicks (Shane, 1996). In Australia, commercial live vaccine grown in chick
embryo liver cells containing the FAdV-8b strain is applied for vaccination of broiler
breeders (Grimes, 2007). Effective protection of the progeny of chickens against IBH-
HHS is achieved by dual vaccination of breeders for FAdV-4 and CIAV (Toro et al.,
2002).
2. Adenoviruses 2.1. Taxonomy
The family Adenoviridae comprises five genera. The members of the genus
Mastadenovirus infect only mammals. The genus Aviadenovirus infects only avian
species; the genus Atadenovirus infects a broad host range such as ruminants, marsupials,
reptiles, and birds, while the genus Siadenovirus infects both amphibian and avian
species. Recently added to the family is the genus Ichtadenovirus. Virus was isolated
from the white sturgeon (Kovács et al., 2003). The genome of Atadenovirus has very
high (>60%) AT content, while Siadenovirus contains a seemingly unique gene, a
sialidase gene homolog at the left-hand side of the genome (Benko et al., 2005). Each
genus is serologically distinct from other genera and also differs significantly in genome
15
organization (Benko et al., 2005). The genus Aviadenovirus, also referred to as group I
avian adenoviruses, comprises five species of FAdVs with one or more serotypes in each.
Species in the genus Aviadenovirus are classified as Fowl adenovirus A (FAdV-1); Fowl
adenovirus B (FAdV-5); Fowl adenovirus C (FAdV-4, FAdV-10); Fowl adenovirus D
(FAdV-2, FAdV-3, FAdV-9, FAdV-11); Fowl adenovirus E (FAdV-6, FAdV-7, FAdV-
8a, FAdV-8b); Goose adenovirus GoAdV-1, GoAdV-2, GoAdV-3; and Falcon
adenovirus A FaAdV-1. Species designation depends on several criteria, such as
calculated phylogenetic distances, restriction fragment length polymorphism (RFLP),
host range, pathogenicity, cross neutralization, and ability to recombine. The first
molecular classification of fowl adenoviruses was based on RFLP of the DNA given by
BamHI and HindIII (Zsák and Kisary, 1984).
2.2. Characteristics of adenoviruses 2.2.1. Structure and physicochemical composition of the adenovirus
The members of the family Adenoviridae are non-enveloped viruses with regular
icosahedral capsids that are 70-90 nm in diameter and have a pseudo T number of 25
(Stewart et al., 1991). The key structural component of adenoviruses is a homotrimeric
hexon (Fig. 1.1.). There are 240 hexons on the faces and edges of the capsid. The pentons
lie at the 12 vertices of the icosahedral capsid and comprise penton bases and protruded
fibers (van Oostrum and Burnett, 1985). The hexon, a trimer of protein II, has the largest
mass and methionine content in the virus structure. The DNA sequence revealed 28
methionine residues among the 967 amino acids (aa) of protein II (Akusjarvi et al., 1984;
Jornvall et al., 1981). The penton consists of the penton base, a pentamer of polypeptide
16
III, and the fiber, which is a trimer of polypeptide IV. The fiber is formed by interaction
of three polypeptide IVs, noncovalently bound into a multimeric structure (Hong and
Engler, 1996). The complete adenoviral structure was determined by cryoelectron
microscopy and three-dimensional image reconstruction methods in 1991 (Stewart et al.,
1991). Adenoviruses contain 13% DNA and 87% protein. The virus has no membranes or
lipids and hence is stable in solvents such as ether and ethanol (Hong and Engler, 1996).
Virions can be disrupted with 5 M urea, 10% pyridine, acetone, or multiple freeze-thaw
cycles (Maizel et al., 1968a). Cesium chloride densities from 1.32 to 1.37 g/ml have been
described, presumably due to differences in DNA contents (Adair and Fitzgerald, 2008).
2.2.2. Proteins
The viral capsid contains the structural proteins II, III, IIIa, IV, VI, VIII, and IX
(Fig. 1.1). Proteins V, VII, X, and terminal protein (TP), which directly interact with the
viral DNA, form the viral core (Hosokawa and Sung, 1976; van Oostrum and Burnett,
1985; Lehmberg et al., 1999). The polypeptides present in the mature virion are
designated by Roman numerals from II to XII in order of their decreasing molecular mass
in acrylamide gels (Maizel et al., 1968b). Protein II (hexon protein), 3 of which form a
hexon, is the most abundant protein in the virion and defines the type, group, and
subgroup antigenic determinants (Norrby, 1969). Protein III (penton protein) has diverse
functions, including pentamerization to form a penton base, and ensure stable fiber-
penton base interaction (Karayan et al., 1997). Protein IV (fiber protein) interacts with
the penton base at the vertices of the icosahedral capsid. The fiber is directly responsible
for viral attachment and internalization of the virus into the host cell (San Martin and
17
Burnett, 2003). Commonly, the fiber structure is divided into three distinct regions
(Green et al., 1983). The first region consists of the N-terminus, also known as the tail,
which is embedded into the penton base. The second region, designated as the shaft,
contains numerous repeating motifs of approximately 15 aa. The third region located at
the C-terminus, the end of the shaft, is called the knob and is necessary and sufficient for
virion binding to the host cell. The mammalian adenoviruses, with a few exceptions
(HAdV-40 and HAdV-41), have one fiber. However, FAdVs have 2 fibers (Gelderblom
and Maichle-Lauppe, 1982) of similar length except in the chicken embryo lethal orphan
(CELO) virus, FAdV-1, in which the fibers are of different lengths. The long fiber of
FAdV-1 is four times longer than the short one and is placed at a 90º angle (Hess et al.,
1995). The differences in the FAdV-1 fibers might be responsible for some of the
properties of the virus, such as hemagglutination and abundant growth in embryonated
eggs that other fowl adenoviruses do not have.
Minor capsid components, including proteins IIIa, VI, VIII, and IX complete the
capsid structure and primarily act as cement proteins. The location of each of them and
their precise role in the virus life cycle has yet to be fully understood. The location of
polypeptide IIIa has been recently defined as a position below the penton base and this
protein is associated with others at the vertex, mainly with the hexon and protein V
(Saban et al., 2005). The location of polypeptide VI in the virion appears to be inside
cavity of every hexon trimer, connecting the bases of two adjacent peripentonal hexons
and tethering the capsid to the core region (Silvestry et al., 2009). Protein VI is liberated
from the virion when the acidic environment of the endosome triggers conformational
18
changes, and this promotes membrane disruption to allow the transport of the virus to the
nucleus (Wiethoff et al., 2005). Moreover, protein VI harbors a PPxY motif involved in
reapid microtubule-dependent intracellular movement and infectivity. Inactivation of the
PPxY motif leads to post-entry delay with consequence on reduced infectivity and
prevent efficient accumulation of the virions at the microtubule organizing center
(Wodrich et al., 2010; Maier and Wiethoff, 2010).
Polypeptide VIII is located at the inner surface of the capsid and it is bound
between the peripentonal hexons and the rest of the capsid (Stewart et al., 1993). The
smallest minor capsid protein, protein IX, is present as a trimer and helps to stabilize the
virion, as mutant virions lacking this protein are less stable than the wild type virus
(Colby and Shenk, 1981). Proteins V, VII, µ, and TP directly interact with the linear
double-stranded DNA to form the viral core (van Oostrum and Burnett, 1985; Lehmberg
et al., 1999). Polypeptide VII is a major core protein with over 800 copies per virion and
together with DNA forms compact repeating structures called ‘adenosomes’ (Tate and
Philipson, 1979). Two copies of the TP are covalently linked to the 5’ end of the genome.
TP acts as a primer for DNA replication and may facilitate circularization of the virus
genome. The TP precursor, pTP, is cleaved by virus-encoded protease at two sites,
creating TP, that is essential for virus replication (Webster et al., 1997).
About 30 adenovirus non-structural proteins have been described (San Martin and
Burnett, 2003). They are generally produced in small quantities and the precise role for
most of them is still unknown. To date, the structures of two of the non-structural
19
proteins have been described, DNA-binding protein (DBP) and viral protease (Tucker et
al., 1994; McGrath et al., 2003). DBP is a multifunctional nuclear phosphoprotein. It is a
major product of early region E2A and is essential for viral DNA replication. It is also
required for the control of early and late transcription (Ward et al., 1998; van der Vliet et
al., 1978). This protein has two domains: a highly phosphorylated N-terminal domain of
173 aa that contains a nuclear localization signal and the C-terminal domain of 356 aa
(Morin et al., 1989). The highly conserved and non-phosphorylated C-terminal domain
binds DNA and is active in DNA replication (Klein et al., 1979; Linne and Philipson,
1980). DBP binds single-stranded (ss) and dsDNA as well as RNA. DBP binds
cooperatively to ssDNA, protecting it against nuclease digestion and destabilizing the
double helix during the elongation phase of DNA replication. Binding to dsDNA occurs
in a non-cooperative fashion and is less stable (Tsernoglou et al., 1984; Zijderveld and
van der Vliet, 1994).
Proteinases play essential roles at various stages of viral replication, including
assembly and maturation of virions. Adenoviruses encode an endopeptidase that is crucial
to the assembly of the virus in the nucleus of infected cells (Weber, 1995). Between 10
and 30 molecules of the protease are packaged into each virion. Cotten and Weber (1995)
demonstrated that the adenovirus-encoded 23K protease is required for cleavage of virion
precursor proteins.
20
The adenoviral enzyme is the only protease known to require DNA for maximum
activity. The atomic structure of the HAdV-2 protease revealed an ovoid-shaped particle
with α-helical regions at both poles and a central β-sheet (Ding et al., 1996).
2.2.3. Genome
The mastadenovirus genome is around 36 kilobase (kb) and consists of four early
regions (E1 to E4), and five late regions (L1 to L5), while the FAdV genome is around 45
kb and is the largest genome among members of the family Adenoviridae (Chiocca et al.,
1996; Ojkic and Nagy, 2000). So far, the complete nucleotide sequences of the genomes
of FAdV-1, FAdV-9, FAdV-8, FAdV-4, and turkey adenovirus type 1 (TAdV-1) are
determined (Chiocca et al., 1996; Ojkic and Nagy, 2000; Grgic et al., 2011; Griffin and
Nagy, 2011; Kajan et al., 2010). The nucleotide sequences at the left and right ends of the
genomes of FAdVs representing species C (FAdV-4 and FAdV-10), D (FAdV-2), and E
(FAdV-8) are also determined and compared to FAdV-1 (FAdV-A) and FAdV-9 (FAdV-
B) (Corredor et al., 2006; Corredor et al., 2008). Nucleotide sequence homology and aa
sequence identities are the highest among members of the same species group, while
different degrees of variations are present among all FAdVs. Moreover, all the studied
FAdVs have a similar gene arrangement, suggesting probably similar gene functions
(Corredor et al., 2008). Interestingly, at the right end of all analyzed FAdV genomes,
homologues to known ORFs such as lipase and Gam-1 are also found (Corredor et al.,
2008). GAM-1, encoded by ORF8, is highly conserved (82%-95.7%) among viruses
belonging to the same species group. In the following text, interesting features related to
FAdVs will be pointed out.
21
FAdVs contain large genomes [FAdV-4 45,667 base pair (bp); FAdV-9 45,063
bp; FAdV-8 44,055 bp) (Griffin and Nagy, 2011; Ojkic and Nagy, 2000; Grgic et al.,
2011] although genes encoding a few structural proteins such as V and IX, and early
regions E1, E3, and E4 described in mastadenoviruses are missing from them. The
genomes of atadenoviruses and siadeoviruses are shorter. For example, the EDSV and
HEV are 33.2 and 26.3 kb, respectively (Hess et al., 1997; Pitcovski et al., 1998). The
highest degree of conservation in the adenovirus genome is in the middle part, which
encodes the structural proteins, whereas the two ends, containing mainly regulatory early
regions, exhibit a larger variation (Davison et al., 2003). In mastadenoviruses, early
regions E1A and E1B are present without exception, while in atadenoviruses, a p32K
gene is present in the position of the E1A of mastadenoviruses. The p32K gene is unique
to atadenoviruses and has been identified in every member of the genus Atadenovirus
studied so far. In addition, only the atadenoviruses seem to have some degree of
homology with the mastadenovirus E1B genes, namely the 55K protein. Moreover, at the
left-hand end of the siadenovirus genome, there is a putative sialidase gene in the place of
the E1 region of mastadenoviruses (Benko et al., 2005). Protein IX and V, found in
mastadenoviruses, are absent in the avi-, at- and siadenoviruses, suggesting that the
virions of these adenoviruses are different structurally. The E3 region seems to exist only
in the mastadenoviruses and no homologous genes were detected in any other genera.
Apparently, E3 represents one of the most commonly used insertion sites by the
adenovirus gene delivery system (Russell, 2000). However, the putative E3 region of
siadenoviruses has no homology with the E3 region of mastadenoviruses. Furthermore,
the right-hand end of the genome harbors an E4 region where the single gene for the 34K
22
protein of mastadenoviruses has its homologue in atadenoviruses only (Benko et al.,
2009). The presence of tandem repeats (TR) at the right end of the genome is reported for
several FAdVs, namely FAdV-9, FAdV-8, FAdV-4, and FAdV-10 (Ojkic and Nagy,
2000; Corredor et al., 2008; Grgic et al., 2011; Griffin and Nagy, 2011). TR-2 of FAdV-
9, consisting of 13 repeats of 135 bp each, is dispensable for virus replication in vitro and
in vivo (Ojkic and Nagy, 2000; 2001). The role of these tandem repeats remains
unknown. The GAM-1 protein is unique to FAdVs only and has functional equivalence to
the human adenovirus E1A 243R, E1A 289R, and E1B 19 kDa proteins. GAM-1 of
FAdV-1 virus is involved in increasing cellular transcription by blocking pRb and
activating E2F-dependent transcription (Lehrmann and Cotten, 1999). Additionally,
lipase ORFs detected in the genome of FAdV-D and FAdV-E species have high identities
to lipase from pathogenic avian herpesviruses (Corredor et al., 2008). Lipase might be
important in virus replication because mutant Marek’s disease ( MDV-1) virus with
foreign gene insertion in the v-Lip LORF2 coding region showed reduced lytic replication
in vivo (Kamil et al., 2005).
23
Figure 1.1. Schematic transection of the adenovirus particle. Taken from Nemerow et al. (2009).
24
2.3. Adenovirus replication cycle 2.3.1. Attachment and penetration
Adenovirus entry into cells involves attachment of the fiber knob to the primary
receptor. Interaction of the penton base with a secondary receptor is responsible for virus
internalization. The virus enters the cell through receptor-mediated endocytosis in a
clathrin-coated vesicle and is transported to endosomes where acidification results in
partial disassembly of the capsid (Fig. 1.2). The altered virion escapes into the cytoplasm
and is transported to the nucleus, where replication occurs (Meier and Greber, 2004).
However, the current model of adenovirus entry is based mainly on studies with HAdVs
of species B and C.
The Coxsackievirus and adenovirus receptor (CAR) is the adenovirus receptor for
most but not all HAdVs (Bewley et al., 1999). CAR is a member of the immunoglobulin
superfamily and comprises two immunoglobulin-like extracellular domains. This receptor
is localized in specialized tight junctions near the tops of the cells and along the
basolateral membranes (Cohen et al., 2001). It is present in adherens junctions along the
lateral sides of airway cells (Walters et al., 2002). In humans, CAR is abundantly
expressed in a number of organs, including the heart, brain, pancreas, and intestines
(Tomko et al., 1997), as well as the lung, liver, and kidney (Fechner et al., 1999). The
presence of CAR on target cells permits virus attachment by HAdVs of species A, C, E,
and F, and also serves as a receptor for several animal adenoviruses: chimpanzee CV-68,
canine type 2, and FAdV-1 (Cohen et al., 2002; Soudais et al., 2000; Tan et al., 2001).
Some HAdVs of species B attach to the CD46 membrane cofactor protein (Cole et al.,
1985), whereas several species D viruses infect cells after attachment to α (2-3) linked
25
sialic acid, a common carbohydrate component of glycoproteins and glycolipids
(Burmeister et al., 2004). Differences in primary receptors are reflected in virus tropism.
CAR-binding serotypes are mainly respiratory viruses, whereas HAdV-40 and HAdV-41
are enteric and their primary receptors are unknown. The main function of the fiber
receptor is to hold the virion in close proximity to the cell surface, permitting interaction
with an integrin molecule. The penton base protein is responsible for virus
internalization, through interaction of an RGD sequence with cell integrins. The RGD
peptide is localized in the flexible loop region of the penton base and serves as a
recognition site for several cellular integrins (Stewart et al., 1997). It has been shown that
the integrins that facilitate adenovirus entry include the vitronectin receptors αvβ3 and
αvβ5 (Wickham et al., 1993), as well as αvβ1 (Li et al., 2001), α3β1 (Salone et al., 2003),
α5β1 (Davison et al., 1997), and all of them recognize RGD ligands. The penton base-
integrin interaction induces activation of PI3 kinase (PI3K) (Li et al., 1998a), p130CAS (Li
et al., 2000), and Rho GTPases (Li et al., 1998b) that are important for virus
internalization and virus escape from the endosome. HAdV-40 and HAdV-41 lack the
RGD motif which explains why different receptors recognize these viruses; instead, the
HAdV-40 carries RGAD and HAdV-41carries IGDD (Albinsson and Kidd, 1999;
Davison et al., 1993). Interestingly, FAdVs also lack an RGD motif (Hess et al., 1995).
26
Figure 1.2. Reproductive cycle of human adenovirus type 2. The virus attaches to a permissive cell and enters the cell via endocytosis 1), 2), partial disassembly takes place prior to entry of particles into the cytoplasm 3). Further uncoating occurs and viral genome is imported into the nucleus 4). Transcription of E1A gene by host cell RNA polymerase II 5). Export of E1A mRNAs to the cytoplasm 6). Synthesis of E1A proteins 7), and import into the nucleus 8), where regulate transcription of both cellular and viral genes 9a). Transcription of the VA genes by host cell RNA polymerase III 9b). The early pre-mRNA species are processed, exported to the cytoplasm 10) and translated 11). These early proteins are imported into the nucleus 12) and cooperate with cellular proteins in viral DNA synthesis 13). Replicated viral DNA molecules serve as template for replication 14) or for transcription of late genes 15). Processed late mRNA species are selectively exported from the nucleus 16). Their efficient translation in the cytoplasm 17) requires the major VA RNA, VA-RNA I and late L4 100kDa protein. The later protein serves as chaperone for assembly of hexons 18). Within nucleus capsids are assembled 19). Assembly requires a packaging signal located at the left of the genome and mature virions are formed when precursor protein are cleaved 20). Progeny virions are released 21). Taken from Flint et al., 2004.
27
2.3.2. Activation of early viral genes
Replication cycle of adenoviruses is determined by the expression of early and
late genes, separated by the onset of viral DNA replication. The E1A is first
transcriptional unit expressed after infection. Initially, transcripts are processed into two
mRNAs that encode proteins 243R and 289R, while three additional alternatively spliced
mRNA congregate later in the infectious cycle (Yang et al., 2002). Furthermore, E1B
gene promoter requires readthrough transcription originating from the upstream E1a
gene, activates transcription factors necessary for transcription of the E1B genes (Shen
and Spector, 2003). Alternative splicing of the E1B pre-mRNA give a rise to E1B 55 kDa
and E1B 19 kDa proteins.
Two E1A proteins are expressed from differentially spliced mRNAs at 1-2 hours
post-infection (h.p.i.) (Yang et al., 2002). Role of these proteins is to promote cell cycle
progression to favor viral DNA replication, counteract the innate immune response
mediated by interferon and inhibit apoptosis. The large 289R protein activates
transcription of early genes from the E1B, E2 and E4 promoters in the infected cells
(Bandara and La Thangue, 1991). E1A proteins interact with cellular proteins and
modulate their function in order to activate the transcription of viral genes. Both 243R
and 289R E1A proteins interact with pRB/p130, TATA-binding protein (TBP),
p300/CBP (Arany et al., 1995), STAT (Look et al., 1998), p21 (Keblusek et al., 1999),
cyclins A and E/CDK complexes (Faha et al., 1993), etc. Binding of the E1A proteins to
Rb bypass the normal regulatory signals and release E2F proteins (Whyte et al., 1988;
28
Chellappan et al., 1992) whose target genes are involved in cell cycle progression and
apoptosis.
2.3.3. DNA replication
Genome replication depends on three viral proteins encoded by the E2
transcription unit: pTP, DNA polymerase, and DBP. These three proteins with the core
origin can initiate DNA replication, but only inefficiently. Additionally, cellular nuclear
factor I (NFI) or CAAT transcription factor (CTF1) and nuclear factor III (NFIII) or
octamer-binding protein (Oct-1) are required for an efficient replication (de Jong and
Van der Vliet, 1999). Origins of DNA replication are present in both inverted terminal
repeats (ITR) of the genome. These origins contain a triplet repeat at genome termini
(GTAGTA) in HAdV-2 and HAdV-5, followed by a conserved region that serves as a
core origin where the pTP/polymerase complex binds. Upon initiation, DNA polymerase
and pTP form a heterodimer and DBP binds dsDNA to coat the entire genome. The
coating of DNA template increases the affinity of NFI for its recognition site in the origin
of replication. Distal to the NFI binding site is the DNA recognition site for NFIII (de
Jong et al., 2003). A pre-initiation complex is then formed and stabilized by direct
interaction of DNA polymerase with NFI and DBP bound to its recognition site (bp 23-
38) in the origin of replication (Armentero et al., 1994). The initiation of adenovirus
DNA replication occurs by a protein-priming process in which the first nucleotide
(dCMP) becomes covalently bound to a serine residue in the viral pTP. When the third
nucleotide is incorporated, the resulting trinucleotide complex (pTP-CAT) jumps back
three bases to the first GTA and disassociation of pre-initiation complex begins (de Jong
29
et al., 2003). The pTP dissociates from DNA polymerase and the latter replicates the
genome by strand displacement. The displaced DNA strand forms a pan-handle structure
by complementary terminal sequences in the ITRs and the complex pTP-DNA
polymerase assembles to initiate the synthesis of the daughter strand (Evans and Hearing,
2002).
2.3.4. Late gene expression
Following the onset of viral DNA replication, the IVa2 and IX genes are
expressed at high levels to activate transcription through MLP (Lutz and Kedinger, 1996;
Lutz et al., 1997). Primary late transcript is about 28 kb in length and gives rise to 5
transcripts termed L1 to L5 by the use of 5 alternative polyadenylation signals. Following
that, each transcript undergoes alternative splicing to generate mature mRNAs for late
proteins (Chow et al., 1980). All alternatively spliced mRNAs contain a 5’ leader
sequence of 200 nts, which comes from the tripartite leader sequence. Leader sequence is
important for efficient translation of late mRNAs independent of host initiation factor
eIF4F (Dolph et al., 1988; Huang and Schneider, 1991).
2.3.5. Assembly and release
Onset of viral DNA replication coupled with the production of large quantities of
structural proteins provides an optimal setting for virus assembly. The assembly of
trimeric hexon capsomeres requires the L4 100K protein (Hong et al., 2005). The 100K
protein is required for the transport of newly synthesized hexon monomers to the nucleus
(Cepko and Sharp, 1982) and also acts as a scaffold to facilitate assembly of hexon
30
trimers (Morin and Boulanger, 1986). Penton capsomeres, consisting of a pentameric unit
and trimeric fiber, assemble slowly in the cytoplasm. Following their production, hexon
and penton capsomeres are imported into the nucleus and virion assembly occurs. The
viral DNA is subsequently encapsidated into an empty capsid via a packaging signal
located at the left end of the viral genome (between nts 194 and 382 in HAdV-5) in a
polar, left-to-right fashion (Schmid and Hearing, 1995; Hammarskjold and Winberg,
1980). Proteins IVa2 and IX are important for packaging of full-length genomes into the
capsid (Ghosh-Choudhury et al., 1987; Zhang and Imperiale, 2003). The core proteins are
encapsidated along with the viral genome. During the maturation process, precursor
proteins pIIIa, pTP, pVI, pVII, pVIII, and pX are cleaved by AdV protease. The cleavage
of precursor proteins is required for the stabilization and infectivity of the viral particles
(Weber, 1995; Cotten and Weber, 1995). The adenovirus death protein (ADP), encoded
by the E3 region, is expressed at high levels late in infection and promotes the release of
virions from infected cells (Tollefson et al., 1996). The mature virions are released by
cell lysis.
3. Avian immune response
In order to understand the immunology of the lymphoid system, it is necessary to
know its basic structure. A number of morphologically and functionally different organs
and tissues have various functions in the development of immune responses. Based on
function, they can be differentiated as primary and secondary lymphoid organs. Thymus,
bursa, and bone marrow are considered primary lymphoid organs, while the secondary
lymphoid organs are lymph nodes, spleen, mucosal-associated lymphoid tissue (MALT),
31
and gut-associated lymphoid tissue (GALT). The thymus-derived cells are a separate
lineage of antigen-specific lymphocytes that do not produce immunoglobulins. The
haematopoietic component that differentiates in the thymus into T lymphocytes,
macrophages, and medullary dendritic cells is derived from the mesoderm (Cooper et al.,
1966). On the other hand, the bursa of Fabricius is a lymphoepithelial organ in birds that
arises as a dorsal endomesodermal diverticulum of the cloaca. Lymphocytes which
differentiate in the bursa of Fabricius do not originate from the bursa rudiment itself, but
are derived from stem cells that invade the bursa between day 8 and 14 of embryonic
development (EID). Additionally, immunoglobulin diversity in chickens is generated by
somatic gene conversion (Ratcliffe and Jacobsen, 1994).
Several important characteristics differentiate innate from adaptive responses.
Firstly, pathogen recognition in the innate system is mediated by elements encoded in the
germline DNA, while B and T cell receptors are formed by rearrangement of receptor
gene elements, a process that provides large diversity of exquisitely different receptors.
Secondly, the different responses of innate and adaptive immunity are a direct
consequence of the diversity and frequency of receptors utilized to sense the pathogens. If
the frequency of the receptor is high, the response can be rapid, but specificity is low,
whereas with B and T cell receptors, which are clonally expressed, the rate of the
response is slow, but specificity is very high. Thirdly, immunological memory makes the
adaptive response more efficient at the second exposure, while the innate response is not
influenced by pre-exposure (Iwasaki and Medzhitov, 2004).
32
3.1. Innate immune response
Innate immunity is based on physical and chemical barriers of infection, including
different cell types responsible for recognizing invading pathogens and activating
immune responses. The cellular components include antigen-presenting cells (APCs),
dendritic cells (DCs), phagocytic macrophages and granulocytes, cytotoxic natural killer
(NK) cells, and γδ T lymphocytes. The first response recognizes microorganisms via
germline-encoded pattern-recognition receptors (PRRs) (Akira et al., 2006). These
receptors are capable of recognizing invading pathogens by two mechanisms. Firstly, the
PRRs recognize microbial components via pathogen associated molecular patterns
(PAMPs), which are essential for the survival of the microorganism and are conserved
within a group of pathogens. For example, lipopolysaccharide and double-stranded RNA
(dsRNA) are PAMPs associated with Gram-negative bacterial infections and viral
infections, respectively. The second way for receptors to sense infection is indirect, via an
encounter between PRRs and host factors, when the latter are present in unusual locations
as a direct consequence of infection, inflammation, or other types of cellular stress.
Examples of such events are host-derived intracellular heat-shock proteins (HSPs) that
are recognized by some PRRs when exposed to the extracellular environment (Beg,
2002). The main function of PRRs comprise enhancement of phagocytosis, activation of
complement cascades, production of inflammatory cytokines, and maturation of DCs,
that all together orchestrate the early host response to infection and are an important link
to the adaptive immune response (Akira et al., 2006).
33
Toll-like receptors (TLRs) as pattern-recognition receptors are most extensively
studied and were originally identified as a gene product essential for dorso-ventral
patterning during embryogenesis as well as for the antifungal response in Drosophila
(Lemaitre et al., 1996). Structurally, TLRs are type I integral membrane glycoproteins
characterized by an extracellular ligand-binding domain containing leucine-rich repeat
(LRR) motifs and a cytoplasmic signaling domain homologous to that of the interleukin 1
receptor (IL-1R), named the Toll/IL-1R homology (TIR) domain (Bowie and O’Neill,
2000). Pathogen binding to TLRs through PAMP-TLR interaction causes receptor
oligomerization which triggers intracellular signal transduction (O’Neill and Bowie,
2007). To date, a total of 13 TLRs (TLR1 to TLR13) have been identified in mammals
and mice, and each recognizes distinct PAMPs derived from different microbial
pathogens, including viruses, bacteria, fungi, and protozoa (Kaisho and Akira, 2006).
Temperley et al. (2008) found a total of ten TLRs in chicken, with only five human
orthologs, one fish ortholog, and three unique to chickens. Chromosome mapping of
chicken TLR genes shows that half of theTLR genes are located on chromosome 4
(Temperley et al., 2008). Commonly, TLRs separate into subgroups expressed at the cell
surface (TLRs 1, 2, 4, 5, 6, and 10) or at endosomes (TLRs 7, 8, and 9). Distribution can
be cell type dependent. TLR3 is expressed on the surface of human fibroblasts but
monocyte derived immature DCs express TLR3 intracellularly and not on the cell surface
(Matsumoto et al., 2003; Wagner, 2004). Usually, TLRs expressed on endosomal
membranes recognize RNA and DNA motifs, whereas cell membrane bound TLRs
recognize proteins, lipoproteins, and lipopolysaccharide.
34
A second large class of PRRs is the family of intracellular sensors of bacterial
motifs known as nucleotide-binding oligomerization domain (NOD) proteins. The NOD
proteins contain an amino-terminal signaling domain and a carboxy-terminal ligand-
binding domain. The ligand binding domain is an LRR domain very similar to that of
TLRs (Inohara and Nunez, 2003).
A third class of PRRs, RNA helicases, were identified by Yoneyama et al. (2004).
The authors demonstrated that two cytoplasmic proteins, retinoic acid inducible gene
(RIG) I and melanoma differentiation associated gene 5, each with two amino-terminal
signaling domains and a carboxy-terminal helicase domain, become activated in response
to virus infection. Moreover, the authors concluded that helicases are probably kept in a
latent state in the absence of ligand engagement, and become activated through a
mechanism dependent on the ATPase activity of the helicase domain (Yoneyama et al.,
2004).
Lastly, the dsRNA-activated protein kinase R (PKR) has emerged as an important
signal transducer in the proinflammatory response to different agents. The activation of
PKR during infection by viral dsRNA down-modulates protein synthesis in virus infected
cells and inhibits viral replication (Nanduri et al., 2000).
In order to understand the cellular responses raised by different viruses, it is
necessary to know which PRRs become activated during viral infection and which viral
molecules serve as ligands for different PRRs. TLRs expressed on the cell surface, such
35
as TLRs 2 and 4, are capable of recognizing viral proteins. Innate sensing of the members
of the herpesvirus, retrovirus, and paramyxovirus families by TLRs has been
demonstrated by Compton et al. (2003) and Rassa et al. (2002). They showed that
detection of measles virus and cytomegalovirus (CMV) particles by TLR2 results in the
activation of innate immune responses. On the other hand, respiratory syncytial virus and
mouse mammary tumor virus are potent triggers of proinflammatory cytokine expression
that occurs via ligation to TLR4.
Moreover, it has been shown by Beg (2002) that host proteins may also activate
the innate immune system during viral infections. Several endogenous proteins activate
the immune system via TLR2 and TLR4 when located extracellularly. Some of them are
HSPs found in all prokaryotes and eukaryotes which under normal physiological
conditions are expressed at low levels. In addition, stress stimuli such as environmental
(UV radiation, heavy metals), pathological (viral, bacterial infection, fever), or
physiological (cell differentiation, growth factors) stimuli can cause a stress response
characterized by increased intracellular HSP synthesis. Asea et al. (2002) showed that
HSP exits mammalian cells and interacts with cells of the immune system, exerting
immunoregulatory effects. The same authors showed that exogenously added HSP70 has
potent cytokine activity, activates NF-κB, up-regulates the expression of
proinflammatory cytokines in human monocytes, and utilizes both TLR2 and TLR4 to
transduce its proinflammatory signal in a CD14 dependent fashion (Asea et al., 2002). It
has been reported by Taddeo et al. (2002) that expression of HSP70 is robustly elevated
during lytic herpes simplex virus infection and TLR2 contributes to HSV-induced
36
inflammation. This result shows a potential role of endogenous ligands in the
inflammatory response raised during lytic viral infection by HSV-1.
As described previously, TLR3, TLR7/8, and TLR9 are expressed primarily in
endosomes and are activated only if the ligands reach this cellular compartment. It has
been shown by Matsumoto et al. (2003) that TLR3 recognizes dsRNA and transduces
signals to activate NF-κB and interferon (IFN)-β promoters. Regulation and localization
of TLR3 are different in each cell type, reflecting participation of cell type-specific
multiple pathways in antiviral IFN induction via TLR3. It is known that dsRNA serves as
an alarm signal associated with viral infection and leads to stimulation of the innate
immune response. In contrast, the immunostimulatory potential of ssRNA is still poorly
understood. Heil et al. (2004) reported that guanosine (G)- and uridine (U)-rich ssRNA
oligonucleotides derived from the human immunodeficiency virus-1 (HIV-1) stimulate
dendritic cells and macrophages to secrete interferon α and proinflammatory as well as
regulatory cytokines. It has been shown that murine TLR7 and human TLR8 mediate
species-specific recognition of GU-rich ssRNA. These results suggest that ssRNA
represents a physiological ligand for TLR7 and TLR8 (Heil et al., 2004).
Recent studies have shown that viral DNA genomes serve as TLR9 ligands for
herpesviruses. Lund et al. (2003) provided the first evidence that plasmacytoid dendritic
cells (pDCs) recognize cytosine-phosphate-guanosine (CpG) motifs present in the
genomic DNA of viruses via TLR9, and that this recognition is required for IFN-α
secretion by these cells. Additionally, TLR9-mediated recognition of HSV suggests the
importance of this recognition pathway for other DNA viruses by the pDCs (Lund et al.,
37
2003). McGuire et al. (2011) showed that HAdV-5 infection induces reactive oxygen
species (ROS) to activate immune cells. ROS induction depends on virally induced
endosomal rupture and release lysosomal cathepsin which leads to mitochondrial
membrane disruption and release of ROS from mitochondria. Moreover, disruption of
lysosomal membranes and the relase of cathepsin B in cytoplasm are essential for HAdV-
5 induced Nod-like receptor (NLRP3) activation (Barlan et al., 2011a, b).
3.1.1. Cytokines
Cytokines are soluble, low molecular weight polypeptides and glycopeptides,
often further subclassified as monokines and lymphokines produced by the
monocyte/macrophages and lymphocytes, respectively. According to Murphy et al.
(2000), a strong cellular immune response against intracellular pathogens is mediated by
IFN-γ-secreting Th1 cells, while Th2 cells, producing IL-4, IL-5, and IL-13, are involved
in responses to parasitic pathogens.
IFN is a glycoprotein produced by white blood cells and viral-infected cells in
response to viral infection. The IFNs are divided into three distinct classes, termed IFN-α,
IFN-β, and IFN-γ. Type I interferons IFN-α and IFN-β are the key cytokines produced
after viral infection and mediate induction of both innate and adaptive immunity to
viruses (Theofilopoulos et al., 2005). They also induce the maturation of DCs, mediate
antigen presentation via major histocompatibility complex (MHC) class I, and mediate
induction of antigen-specific CD8+ T cell responses. Cellular production of IFN-α/β after
virus infection is triggered by dsRNA and virtually any cell type can express IFN-α/β
38
following virus infection. Type II IFN (IFN-γ or immune IFN) is derived from T
lymphocytes and NK cells. It has been shown that IFN-γ has some involvement in most
stages of the inflammatory and immune responses. To date, all three types of chicken IFN
have been cloned (Sekellick et al., 1994; Digby and Lowenthal, 1995). IFN-γ is an
essential cytokine in the host defense against various pathogens. It is directly responsible
for inhibition of MDV replication by inducing nitric oxide (NO) synthesis (Xing and
Schat, 2000), also reducing replication of coccidian parasites and inhibiting src
oncogene-induced tumor growth (Plachy et al., 1999; Gobel et al., 2003).
IL-18 is considered a proinflammatory cytokine expressed by a wide variety of
cells such as macrophages, DCs, and Kupffer cells (Kaiser et al., 2003). It is produced as
a 24-kD inactive precursor (pro-IL-18) that needs to be cleaved by endoprotease IL-1β-
converting enzyme (caspase-1) to generate biologically active IL-18. However, cleavage
of pro-IL-18 is not exclusive to caspase-1: proteinase 3 also can generate biologically
active IL-18 (Gracie et al., 2003). Although originally identified as an inducer of IFN-γ,
IL-18, in combination with IL-12, enhances T and NK cell maturation, cytokine
production, and cytotoxicity (Kaiser et al., 2003). Based on its potent immunomodulatory
properties, IL-18 is essential to host defenses against a variety of infections. During viral
infection, IL-18 effectively induces IFN-γ and activates CD8+ T cells which are important
for viral clearance. In an in vivo model of vaccinia virus infection, administration of IL-
18 significantly suppresses pock formation (Tanaka-Kataoka et al., 1999). Moreover,
clearance of neurovirulent influenza A virus is impaired in IL-18-deficient mice (Mori et
al., 2001). Cho et al. (2001) reported that down-modulation of IL-18-induced immune
39
responses by human papilloma virus (HPV) oncoproteins might contribute to viral
pathogenesis or carcinogenesis. Kaiser et al. (2003) showed differential IL-18 expression
in the spleens of chickens of susceptible and resistant genotypes following MDV
infection.
IL-10 is a regulatory cytokine, originally named cytokine-synthesis inhibitory
factor (CSIF) because of its ability to modulate the immune response towards a type 2
response by inhibiting IFN-γ synthesis (Rothwell et al., 2004). Interestingly, IL-10 can
have both an immunosuppressive and an immunostimulating effect on cell mediators of
adaptive immunity. IL-10 is produced by multiple cell types, including DC, B cells,
macrophages, CD4 T cells, CD8 T cells, and NK cells, as well as innate and adaptive
regulatory T cells (O’Garra and Vieira, 2007). Several authors report that IL-10 acts
directly on antigen presenting cells in order to decrease stimulatory molecule expression
and alter cytokine production (Carbonneil et al., 2004; Moore et al., 2001). It has been
shown by Sloan and Jerome (2007) that herpes virus remodels the intracellular signaling
pathway of T cells to induce production of IL-10 in order to evade the host immune
response. Abdul-Careem et al. (2007) showed that the presence of IL-10 is closely
correlated to vaccine failure in MDV-infected chickens. IL-10 is also a contributing
factor in susceptibility of chickens to Eimeria maxima infection (Rothwell et al., 2004).
Viruses have evolved a number of diverse strategies to avoid the host
inflammatory response. Such strategies have been observed for poxviruses and
herpesviruses, which modulate cytokine action by encoding secreted forms of receptors
40
for cytokines. On the other hand, adenoviruses modulate cytokine expression by
encoding intracellular proteins that counteract TNF-α (Lesokhin et al., 2002; Wold et al.,
1994). Moreover, E1A and E3 gene products down-regulate transcription of IL-8
cytokine (Lesokhin et al., 2002). Zhou et al. (2007) showed that the porcine adenovirus
type 3 E1Blarge protein inhibits the expression of the IL-8 cytokine. A similar observation
has also been reported in vitro in Marek’s disease lymphoblastoid cell line (MSB-1),
where treatment with M. gallisepticum suppresses IL-8 gene expression (Lam, 2004).
However, Booth and Metcalf (1999) reported type-specific induction of IL-8 in human
pulmonary epithelial cells upon infection with HAdV-7. It has been suggested that
Ras/Raf/MEK/Erk pathway is necessary for the HAdV-7 induction of IL-8 in lung
epithelial cells and lung tissue (Alcorn et al., 2001). Wu et al. (2006) showed that HAdV-
7 internalization is calcium dependant and is essential for IL-8 induction following
infection. Additionally, HAdV-7 infection initiate IFN-γ inducible protein 10 (IP-10)
secretions from macrophages and epithelial cells (Wu et al., 2010). IL-8 is a potent
chemotactic cytokine for neutrophils and T lymphocytes and is produced by macrophages
and other cell types such as epithelial cells.
3.2. Adaptive immunity
Immune mechanisms mediated by adaptive immunity are divided in antibody
(humoral) immunity and cell-mediated immunity. Humoral immunity is provided by
antibodies produced in response to antigen exposure. It is specific to an antigen and
comes in two forms: passive and active immunity. Passive immunity may be either
naturally or artificially acquired. Naturally acquired immunity refers to antibody-
41
mediated immunity conveyed to an embryo by its mother during egg formation or via the
colostrum in mammals. Artificially acquired passive immunity is short-term
immunization achieved by the transfer of blood serum containing antibodies, either orally
or through injection. Passive immunity provides immediate protection, but the immune
system is not stimulated and the chick will not produce its own antibodies and does not
develop memory. On the other hand, active immunity is obtained from the chick’s own
immune system and is subdivided into naturally and artificially acquired immunity.
Naturally acquired active immunity occurs through contact with a disease-causing
pathogen, while artificially acquired active immunity is developed through vaccination.
Active immunity has the ability to remember specific pathogens, resulting in quicker and
more effective responses to repeated exposure. Immunoglobulins (Ig), the key proteins of
the specific immune response, are produced by plasma cells and lymphocytes. Their basic
unit structure contains four polypeptide chains, two heavy (H) and two light (L) chains,
that form the monomeric unit (H2L2) (Litman et al., 1993). Using immunocytochemical
and genetic techniques, five classes of mammalian Ig have been identified: IgA, IgD,
IgE, IgG, and IgM, while avian Igs have been classified into three classes: IgA, IgY
(IgG), and IgM. Avian IgA is the predominant form of antibody (Ab) activity in body
secretions. IgY is the major avian Ig with similarities to both mammalian IgG and IgE.
IgY is produced after IgM in the primary antibody response and is main isotype produced
in the secondary response (Warr et al., 1995). Chicken IgM is the first isotype to be
expressed after initial exposure to a new antigen and is structurally and functionally
homologous to its mammalian counterpart (Da Silva Martins et al., 1991).
42
Infection with fowl adenoviruses elicits a strong Ab response against viral
proteins. Chickens infected with FAdVs seroconvert at 1 week p.i. and the highest titers
of anti-FAdV Abs are detected 3 weeks after inoculation (Ojkic and Nagy, 2003; Maiti
and Sarkar., 1997). However, the Ab response depends on the dosage and the route of
inoculation (Ojkic and Nagy, 2003). Moreover, fowl adenovirus-specific Abs are
commonly found in breeder and layer flocks and exposure to multiple serotypes is
reported by several authors (Adair et al., 1980; Adair and Fitzgerald, 2008). Serological
monitoring of Ontario broiler breeder flocks affected with IBH caused by an FAdV-2 at
an early age showed that seroconversion occurs within 4 weeks and birds are resistant to
reinfection with the same serotype 8 weeks after the primary infection (Philippe et al.,
2007).
On other hand, the antigen specific component of the cell-mediated immunity is
the T cell. Different types of T cells are differentiated based on their functional
capabilities and cell surface markers. T cells which play regulatory role in adaptive
immunity are usually referred to as T helper (Th) cells and express CD4 molecules on
their surface. Both avian and mammalian adaptive immunity depend on CD4+ Th cells
(Arstila et al., 1994). However, not much is known about this part of immune response
with respect to adenovirus infection.
43
Hypothesis and objectives 1. Hypotheses
1.1. Hypothesis 1
Viruses representing the fowl adenovirus species group C (FAdV-C) (FAdV-4 field
isolate) and fowl adenovirus species group E (FAdV-E) (FAdV-8 field isolate) are not
primary pathogens.
1.2. Hypothesis 2
Specific sequences of the fiber gene contribute to virulence of FAdVs.
2. Objectives
In the last several decades, IBH has been reported in almost every country around
the world (Philippe et al., 2005; Gomis et al., 2006; Goodwin et al., 1993; Young et al.,
1972; Saiffudin and Wilks, 1991; Erny et al., 1991). Although fowl adenoviruses are
associated with numerous clinical conditions, their primary role in disease is still not
understood. Therefore, my first and second objective were focused on determining the
pathogenicity of two Ontario isolates, one isolated from a flock with clinical signs of
IBH, with IBH as the final diagnosis, while another virus was isolated from a flock
without clinical signs of the disease. Due to an increased interest in adenoviruses as
vaccine and gene delivery vectors, it is important to understand the molecular aspects of
infection and the host responses to them (Corredor and Nagy, 2010b). Therefore, the
following part of the research was focused on cytokine gene expression following FAdV
infection. This is the first study related to FAdV-associated cytokine gene expression and
44
particular cytokines (IFN-γ, IL-18, IL-10, and IL-8) were selected because of their
important roles against intracellular pathogens. IFN-γ is involved in most stages of the
inflammatory and immune response and is the essential cytokine in the host defense
against various pathogens (Sekellick et al., 1994; Digby and Lowenthal, 1995). IL-18 is a
potent inducer of IFN-γ and is important for viral clearance (Kaiser et al., 2003). IL-10 is
a regulatory cytokine which has both an immunosuppressive and an immunostimulating
effect on cell mediators of adaptive immunity (O’Garra and Vieira, 2007). IL-8 is a
potent chemotactic cytokine critical to the process of inflammation (Oppenheim et al.,
1991).
The third objective focuses on fiber protein, a major constituent of the adenovirus
capsid involved in facilitating virus entry. It has been reported that FAdV-8 virulence is
determined by a difference in fiber protein alone (Pallister et al., 1996). Variations in
fiber gene sequences have also been implicated in the differences in tissue tropism and
virulence for the canine adenoviruses (Rasmussen et al., 1995). Yet the role of fiber
protein in the virulence of fowl adenoviruses is not well understood. Therefore, the last
objective examines the possible role of the fiber gene in FAdV virulence by analysis of
sequences from pathogenic and non-pathogenic FAdV-11 and FAdV-8 field isolates.
2.1. To determine the pathogenicity of a fowl adenovirus serotype 8 (FAdV-8) isolate in
specific pathogen free (SPF) layer chickens following oral and intramuscular
inoculations.
45
2.1.1. To sequence and analyze the genome of FAdV-8 and study the relationship with
other fowl adenovirus genomes.
2.1.2. To analyze cytokine IFN-γ, IL-10, IL-18, and IL-8 gene expression in SPF
chickens after infection with FAdV-8.
2.2. To determine the pathogenicity of a fowl adenovirus serotype 4 (FAdV-4) isolate in
SPF layer chickens following oral and intramuscular inoculations.
2.2.1. To analyze cytokine IFN-γ, IL-10, IL-18, and IL-8 gene expression in SPF
chickens after infection with FAdV-4.
2.3. To analyze and compare the fiber gene sequences of FAdV isolates from field cases
in order to determine differences that may contribute to virulence.
46
CHAPTER 2 Pathogenicity and complete genome sequence of a fowl adenovirus serotype 8 isolate
Helena Grgić1, Dan-Hui Yang2 and Éva Nagy1*
1Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, N1G 2W1, Canada. 2Department of Molecular and Cellular Biology, University of Guelph, N1G 2W1 Guelph, Ontario, Canada
*Corresponding author: Éva Nagy E-mail: [email protected] Tel: +1 519 824 4120 ext 54783 FAX: +1 519 824 5930 The GenBank accession number of the sequences reported in this paper is GU734104.
Author’s contributions
HG designed and performed experiment, carried out analysis and wrote the paper. D-HY
provided guidance for qPCR. EN provided guidance during the research and critically
revised manuscript.
Published in Virus Research. 2011. 156:91-97.
47
Abstract
In this study we determined and analyzed the complete nucleotide sequence of the
genome of a fowl adenovirus serotype 8 (FAdV-8) isolate and examined its pathogenicity
in chickens. The full genome of FAdV-8 was 44,055 nucleotides in length with a similar
organization to that of FAdV-1 and FAdV-9 genomes. No regions homologous to early
regions E1, E3 and E4 of mastadenoviruses were recognized. Along with FAdV-9,
FAdV-8 has only one fiber gene and with regard to sequence composition and genome
organization, FAdV-8 is closer to FAdV-9 than to FAdV-1. Moreover, our findings
suggest that FAdV-1 of species Fowl adenovirus A as the current type species despite its
historical priority is not representative of the genus Aviadenovirus, and that FAdV-8 or
FAdV-9 in species Fowl adenovirus E and Fowl adenovirus D, respectively, would be
more suitable for that designation. Additionally, pathogenicity of FAdV-8 was studied in
specific pathogen free chickens following oral and intramuscular inoculations. Despite
lack of clinical signs and pathological changes, virus was found in tissues and cloacal
swabs of all birds with the highest viral copy numbers present in the cecal tonsils. The
highest virus titers in the feces for orally and intramuscularly inoculated chickens were
recorded at day 10 and 3 post-infection, respectively.
Keywords
FAdV-8 complete nucleotide sequence; pathogenesis; virus shedding; highest viral copy
numbers in the cecal tonsils
48
Abbreviations
FAdV-8, fowl adenovirus 8; d.p.i., days post-infection; ORF, open reading frame; pfu,
plaque forming unit; SPF, specific pathogen free
49
1. Introduction
Adenoviruses are in the family Adenoviridae and grouped into five genera. While
mastadenoviruses and aviadenoviruses infect mammals and birds respectively, viruses in
the genera Atadenovirus and Siadenovirus have a broader host range. The genus
Aviadenovirus currently comprises five fowl (Fowl adenovirus A-E), one falcon (Falcon
adenovirus A) and one goose adenovirus (Goose adenovirus) species (ICTV, Virus Taxonomy:
2009 Release). The type species is Fowl adenovirus A and is represented by fowl
adenovirus 1 (FAdV-1) (Benkő et al., 2005).
The first molecular classification of fowl adenoviruses was based on restriction
fragment length polymorphisms (RFLP) of their DNA genome (Zsák and Kisary, 1984).
The genomes of fowl adenoviruses (FAdVs) are larger than that of the human
adenoviruses (HAdVs), in spite of the fact that open reading frames (ORFs) for a few
structural proteins such as V and IX, and early regions E1, E3 and E4 responsible mainly
for host cell interaction and immune modulation in mastadenoviruses, are absent in
FAdVs. The complete nucleotide sequences are available for the genomes of FAdV-1
(CELO virus) and FAdV-9 (A2-A strain) from the species Fowl adenovirus A and Fowl
adenovirus D, respectively. The FAdV-9 genome (45,063 bp) is 1,259 bp longer than
that of FAdV-1 (43,804 bp) (Ojkic and Nagy, 2000; Chiocca et al., 1996). Partial
sequences are also published for the left and right ends of the genomes of FAdV-2,
FAdV-4 and FAdV-10 (Corredor et al., 2006; 2008), and for the late region of FAdV-4
and FAdV-10 (Mase et al., 2010; McCoy and Sheppard, 1997; Sheppard et al., 1998).
Although it is known that the highest degree of conservation among all adenovirus
50
genomes is in the middle region that encodes the structural proteins only a few complete
genome sequences are available for FAdVs.
FAdVs are common infectious agents of poultry although most of them are
subclinical or associated with mild clinical signs (McFerran and Smyth, 2000). However,
some FAdV strains are associated with a variety of diseases, such as inclusion body
hepatitis (IBH) which is diagnosed in almost every part of the world (Adair and
Fitzgerald, 2008). The disease affects mostly 3 to 7 weeks old meat-type chickens
(McFerran et al., 1976; Ojkic et al., 2008a,b), although Bickford (1974) and Hoffmann et
al. (1975) also reported cases in pullets and replacement layers, respectively. IBH
outbreaks are characterized by sudden onset of mortality which usually ranges from 2-
10%. (Adair and Fitzgerald, 2008). The varying degree of mortality correlates to the
pathogenicity of the virus, secondary infections and susceptibility of the chickens. In
Canada, the most commonly isolated FAdVs belong to serotypes 2, 7, 8, and 11 (Ojkic et
al., 2008a,b). Following initial multiplication the virus spreads to virtually every organ,
although the main sites of virus replication are the respiratory and alimentary tracts
(Cook, 1983; Saifuddin and Wilks, 1991; Hess, 2000).
The goals of this study were to (1) determine the complete nucleotide sequence of
the genome of a fowl adenovirus serotype 8 (FAdV-8) isolate from an IBH outbreak
representing species Fowl adenovirus E, (2) investigate its genomic organization and
relationship with other adenoviruses (AdVs) and (3) study its pathogenicity in specific
pathogen free (SPF) chickens.
51
2. Materials and methods 2.1. Virus and cells
The serotype 8 FAdV was isolated by the Animal Health Laboratory (AHL),
University of Guelph from an IBH outbreak that occurred in an Ontario broiler farm. The
virus was plaque purified in our laboratory. Virus propagation and titration were done in
a chicken hepatoma cell line (CH-SAH) as described by Alexander et al. (1998).
2.2. Nucleic acid preparation
CH-SAH cells were infected at a multiplicity of infection (m.o.i.) of 0.5. Cells and
supernatants were collected when an extensive cytopathic effect was seen. Virus
purification and viral DNA preparation were performed as described (Ojkic and Nagy,
2001).
2.3. Cloning, sequencing and sequence analysis
The FAdV-8 DNA was digested with BamHI and the fragments were separated in
0.8% agarose gel. DNA from the desired fragments was extracted and cloned into BamHI
digested pBluescript (SK-). Sequencing was first carried out with universal forward and
reverse primers and then completed by primer walking. Multiple nucleotide sequence
alignments using Vector NTI Advance TM 11 were done to identify the conserved regions.
The sequence data were subjected to BLAST analysis with the GenBank database to
determine the nucleotide sequence homologies and amino acid identities and similarities.
ORFs encoding polypeptides over 50 amino acids were identified by the ORF finder from
Vector NTI Advance TM 11.
52
2.4. Pathogenicity assessment of FAdV-8 in SPF chickens
A trial was conducted to assess the pathogenic potential of the virus. One hundred
white Leghorn SPF chickens were hatched at the Arkell Poultry Station from eggs
obtained from the Canadian Food Inspection Agency (CFIA), Ottawa. The birds were
housed at the University Isolation Unit according to the Animal Care Committee of the
University of Guelph in accordance with the Guide to the Care and Use of Experimental
Animals of the Canadian Council on Animal Care and received food (21% unmedicated
starter ration) and water ad libitum. At 9 days of age the chickens were tagged with wing
bands (Ketchum, ON) and sera were collected from all of them. Ten-day-old chicks were
randomly divided into four groups: Group I (FAdV-8)-oral, Group II (FAdV-8)-
intramuscular (im), Group III (control)-oral and Group IV (control)-im. Groups I and II
had 30 birds each and negative control groups III and IV had 20 birds each. Chicks were
each inoculated with 2 x 108 plaque forming units (pfu) of the virus. The control birds
received PBS. All chickens were re-inoculated at 14 days of age with the same dose of
virus.
The birds were observed daily (3x) for clinical signs. Four chicks from treatment
groups I and II and three chicks from the control groups were drawn randomly for
necropsy at each of 0, 3, 5, 7, 14, 21, and 28 days post-infection (d.p.i.) (except day 0
when only two chicks from the controls were euthanized). The chickens were euthanized,
necropsied, examined for the presence of gross lesions and tissues from thymus, lung,
heart, liver, kidney, bursa of Fabricius and cecal tonsils were collected. Each organ was
sectioned into three portions: one was placed into 10% formalin for histology, the second
53
was stored at -70°C for virology and the third was stored at -70°C for qPCR. Cloacal
swabs were taken from all birds at 0, 3, 5, 10, 14, 21, 28 d.p.i. and their virus titers were
determined in CH-SAH cells. In addition, chickens were bled at weekly intervals, 0, 7,
14, 21, 28 d.p.i. and serum samples were tested for FAdV-specific antibodies (Abs) by
enzyme-linked immunosorbent assay ELISA as described (Ojkic and Nagy, 2003).
2.5. Quantitative real time PCR (qPCR) analysis
To determine the viral load in tissues, real time PCR using SYBR Green as
intercalating dye was developed. The FAdV-8 ORF-1 A/B gene (Corredor et al., 2006)
was used as an indicator for the presence of viral DNA with primers; forward primer:
5’-AAATGGTAAACGCGTGGGATC-3’ and reverse primer:
5’-TTCTCCGTCTCCGATCTGG-3’. The 20 l qPCR reaction containing 1 l (10 pmol)
of each primer, 10 l of QuantiTec SYBR Green I PCR Master Mix (Qiagen), 2 l DNA
and 6 l nuclease free water, was prepared using the computerized automated liquid
handling robotics CAS-1200 (Corbett Research). The qPCR program was first run at 15
min at 95C to activate the Taq polymerase, followed by 40 cycles of 95C for 20 sec,
57C for 15 sec, 72C for 20 sec, with an acquiring step at 75C for 15 sec, using the
Corbett Research Rotor-Gene 6000 System. Quantitative measurement of the PCR
product was carried out by incorporation of the SYBR Green I fluorescent dye. To
establish the standard curves, 10-fold serial dilutions (10-1 to 10-7) of FAdV-8 genomic
DNA starting at 12.2 ng/l (2.4 x 108 copies/l) were made and used as qPCR templates.
Melting curve analysis was performed after each run to monitor the specificity of the
54
PCR products. Negative control using DNA extracted from uninfected chicken and no
template control (NTC) were routinely included in each experiment.
2.6. Statistical analysis
Kruskal-Wallis test was performed to evaluate whether viral copy numbers
differed among tissues. Following statistically significant P-value, pair-wise comparison
was performed between different tissues. P-values of 0.05 were considered to be
statistically significant.
3. Results 3.1. Genome size and organization of FAdV-8
The full genome of FAdV-8 was found to be 44,055 base pairs (bp) with a G+C
content of 58%. The position and size of 46 ORFs with potential to encode polypeptides
of at least 50 amino acid (aa) residues are listed in Supplementary Table 2.1. A schematic
of the genome map is presented in Fig. 2.1. The GenBank accession number of the
sequence reported in this paper is GU734104.
The sequences of the left and right ends of this FAdV-8 isolate were determined
earlier in our laboratory (Corredor et al., 2006; 2008). Briefly, the first 6 kb at the left
terminal region of the genome have no significant sequence homology to known
mastadenoviral sequences either at the DNA or protein level. Homologues to the first 10
ORFs (ORF0 to IVa2) in this region described previously for FAdV-1 and FAdV-9 were
present in the genome of FAdV-8 (Corredor et al., 2006). None of the leftward oriented
ORFs at the right end of FAdV-8 showed homology to identified E4 sequences from
55
other AdVs. The FAdV-8 genome encodes three small ORFs (ORF 38652-39047, ORF 39052-
39486, and ORF 39291-39551) which are homologoues to ORF8 and are unique to FAdVs
only. ORF8 encodes the highly conserved (82-95%) GAM-1 protein found in all fowl
adenoviruses. The FAdV-8 genome has a short region of repeated sequences. The repeat
region consists of two consecutive 51 bp-long units, located within ORF19 at nts 35607-
35708, with no homology to the repeats of FAdV-9 TR-1 and TR-2 (Ojkic and Nagy,
2000; Corredor et al., 2008).
The central portion of the FAdV-8 genome, from IVa2 gene at position nt 6208 on the
left strand toward the fiber gene at position nt 30863 on the right strand showed a similar
organization to the central region of genomes of other adenoviral genera. The % identities
of 17 of those proteins are summarized in Table 2.1. Three proteins crucial for viral DNA
replication, DNA polymerase (DNA pol), terminal protein precursor (pTP) and DNA
binding protein (DBP) encoded by E2 were well conserved when compared with their
FAdV-1 and FAdV-9 counterparts. The percentages of aa identity between FAdV-8 and
FAdV-1 DNA pol, pTP and DBP were 66.2%, 59.8% and 58%, respectively. Protein
sequence identities between DNA pol, pTP and DBP of FAdV-8 and FAdV-9 were
79.5%, 74.3% and 73.7%, respectively. The intermediate gene pIVa2 of FAdV-8 showed
64.9% and 87.3% aa sequence identity to its FAdV-1 and FAdV-9 counterparts, whereas
identity to pIVa2 sequences from other AdVs varied from 73% to 94%. For each FAdV-8
protein the highest % identity was observed with the FAdV-9 homologue. Another
intermediate gene, the gene encoding the minor capsid protein IX that functions as capsid
cement could not be identified in FAdV-8. The FAdV-8 equivalent of early region 3 (E3),
56
if it exists, does not seem to be in the same location as that in mastadenoviruses, between
the protein VIII and fiber protein genes. This intergenic region in FAdV-8 was only 253
nts in length and contained one ORF partially overlapping with the protein VIII gene.
The predicted protein sequence had no identity with any described E3 proteins. However,
this ORF had 62% and 70% identity with U-exon of FAdV-1 and FAdV-9, respectively.
In the FAdV-1 genome there are 476 nts between the pVIII and fiber genes while, in
FAdV-9 there are only 254 nts.
AdV late genes are expressed after the onset of viral DNA replication and encode
mostly structural proteins. A region located between nts 8101 and 8386 had 90% identity
to FAdV-9 major late promoter (MLP) and a TATA box was also identified between nts
8360 and 8365. The late genes of FAdV-8 (IIIa, penton base, hexon, pVI and pVIII) were
in the same order as seen for other aviadenoviruses and mastadenoviruses. The genes for
core proteins pVII and pX were also identified on the FAdV-8 genome whereas
sequences for pV, the third core protein found in mammalian adenoviruses could not be
identified. The hexon protein, the major component of the virion, carries the type, group
and subgroup specific antigenic determinants of FAdVs. Nevertheless, a very high
percent identity was noted for the hexon of FAdV-8, 81.2% and 88.3% with FAdV-1 and
FAdV-9, respectively. The FAdV-8 viral protease essential for virus maturation and
cleavage of the precursors of protein VI, VII, VIII, IIIa, X and pTP showed 39% and 37%
homology to its counterparts in FAdV-1 and FAdV-9 genomes, respectively. The 100k
protein required for assembly of hexon trimers and late viral translation was found to
share 50.9% and 53.7% homology with their counterparts in FAdV-1 and FAdV-9,
57
respectively. FAdV-8 has only one fiber gene, similar to FAdV-9, and aa identity
between these two fibers was 53.4%. On the other hand, protein identity with FAdV-1
short fiber gene was 29% while, with the long one was only 17.4%. (Table 2.1). FAdV-8
fiber protein was comprised of 524 aa and the ORF started at nt 30863. Similar to other
adenovirus fiber proteins, it was organized into three regions: tail, shaft and head. The tail
region was 71 aa including the poly(G) stretch in the tail domain that may also form a
connection between the tail and the shaft without belonging to either. The nuclear
localization sequence (NLS) KRAR of the human adenovirus 2 (HAdV-2) fiber (Hong
and Engler, 1996) was not detected in FAdV-8, although the identified RKRP (residues
17-20) could have an NLS function. In many mammalian adenovirus fibers there is a
conserved VYPY sequence which is probably involved in the interaction with the penton
base. The equivalent sequence in FAdV-8 fiber tail was VYPF (residues 53-56). Because
of the absence of the conserved TLWT in FAdV-8 fiber, the actual start of the head
domain could not be determined with certainty.
The short rightward-oriented virus-associated (VA) RNAs transcribed by RNA
polymerase III and located between the pTP and 52K genes in the genome of most
mastadenovirus species could not be identified for the FAdV-8 genome.
3.2. Pathogenicity of FAdV-8 (clinical signs, gross lesions and histology)
Clinical signs or pathologic changes of inclusion body hepatitis were not seen in
groups of chicks after im and/or oral administration of virus. Histopathological
examination revealed mononuclear cell infiltration, degeneration and necrosis of
58
hepatocytes in only three cases. The classical signs of IBH characterized by intranuclear
inclusion bodies in hepatocytes were not present in any of the inoculated chickens.
3.2.1. Viral genome copy number in tissues
Quantitative real-time PCR was employed to establish viral genome copy
numbers in liver, bursa of Fabricius and cecal tonsil of infected birds. No viral DNA was
detected in any tissues from any groups before inoculation and in mock-infected
chickens. The results over the entire study period are summarized in Table 2.2.
Viral DNA was detected in 95.1% of tissue samples collected at 3, 5, 7 14, 21 and 28
d.p.i. Of the 4.9% of tissue samples that were negative all were from the bursa of
Fabricius. Table 2.2 shows the arithmetic means of viral genome copy numbers of these
birds in each group of birds inoculated by different inoculation routes.
The results of the Kruskall-Wallis test indicated significant differences in orally
inoculated birds (P<0.001) and in im-inoculated birds (P=0.0012), and the im-inoculated
group of chickens had higher viral copy numbers. The cecal tonsil was the organ with the
highest number of viral copies through the study period irrespective to inoculation route
followed by liver and the bursa of Fabricius.
3.2.2 Virus titers in feces
No virus was present in cloacal swabs in any groups of chickens before
inoculation and in the mock infected group at all time. The virus titers in feces are given
in Table 2.3. The results showed that the difference in titers between chickens inoculated
orally and im was not statistically significant (P=0.88), when it was tested by 2-sample
59
Wilcoxon test. The highest virus titer in the orally inoculated group was found at day 10
p.i. (1.6x104) while in the im inoculated group it was at day 3 p.i. (3.1x104). Virus titer
began to decline in the orally inoculated group after 10 d.p.i. while, in the im group
decline in virus titer was found after 3 d.p.i. Chickens shed the virus through the entire
study period in both oral and im group.
3.2.3. Antibody response
Abs against FAdV-8 were not detected in any groups before inoculation (0 d.p.i)
and in the mock-infected group at all times. Ab response to viral proteins appeared at 14
d.p.i. with significant differences between inoculated groups and negative control. Ab
titer was slightly lower at 21 d.p.i. but difference in titer between inoculated groups and
negative controls was significant. The differences in titers between birds inoculated im
and orally were significant (P<0.001). Chickens inoculated im had higher titers than
chickens inoculated orally (Fig. 2.2).
4. Discussion
We determined and analyzed the full genome sequence of a FAdV-8 isolate, the
first virus of species Fowl adenovirus E. So far the complete genome nucleotide
sequences of only FAdV-1 (CELO virus) and FAdV-9 (strain A-2A) have been reported
(Chiocca et al., 1996; Ojkic and Nagy, 2000). Nucleotide sequences of the right and left
ends of FAdV genomes representing viruses of four species groups had already been
determined and compared to those of the sequences of FAdV-1 and FAdV-9 (Corredor et
al., 2006; 2008).
60
The genome of FAdV-8 was 44,055 nt in length, with a G+C content of 58% compared
to 54% for FAdV-1 and 54.3% FAdV-9. There were 46 ORFs with potential to encode
polypeptides that were at least 50 aa residues long. Based on this analysis the FAdV-8
genome is 251 nt longer than that of FAdV-1 and 1008 nt shorter than that of FAdV-9
genome. There are several reasons for such differences in size. First, two sets of tandemly
repeated sequences (51 bp long) identified within ORF19 in the FAdV-8 genome, were
much shorter than the repeat TRs found in FAdV-9. The FAdV-9 genome contains two
blocks of tandem repeats located at the right end of the viral genome and are incorporated
within a region rich in ORFs. The shorter region (TR-1) contains five 33 bp long repeats
while the longer region (TR-2) contains thirteen 135 bp long repeats (Cao et al., 1998;
Ojkic and Nagy, 2000). The function of these repeats is unknown, although it has been
reported that the longer subunit is dispensable for in vitro virus replication (Ojkic and
Nagy, 2001). Interestingly tandemly repeated sequences have also been documented in
vaccinia virus and Herpes simplex virus type 1 (HSV-1) (Baroudy and Moss, 1982;
Umene et al., 1984.) Secondly, the short, partially double-stranded VA RNAs transcribed
by RNA polymerase III involved in translational control and inhibition of the interferon
response (Mathews and Shenk, 1991) were absent in the FAdV-8 genome and a similar
finding was reported for the FAdV-9 genome (Ojkic and Nagy, 2000). Currently
identified VA RNAs of mammalian AdVs are transcribed rightward, and human AdVs
contain one or two VA RNA genes located between the pTP and 52k ORFs. FAdV-1 and
EDS viruses have only one VA RNA gene, which is much shorter and located near the
right end of the genome. Although they share a similar position on the genome, the
FAdV-1 VA RNA gene is transcribed leftward which is opposite to its EDSV counterpart
61
(Hess et al., 1997; Chiocca et al., 1996). Thirdly, the complete DNA sequence of FAdV-
1 revealed that FAdV-1 is the only fowl adenovirus sequenced to date with two fiber
genes. The actual fiber ORF sizes differed greatly in length, 793 and 412 aa, respectively
(Hess et al., 1995; Chiocca et al., 1996). In contrast to FAdV-1, only one fiber gene was
identified in the corresponding region of the FAdV-8 genome. The fiber protein is the
major constituent of the outer capsid involved in virus entry into permissive cells.
Interestingly, an equivalent sequence motif in the FAdV-8 fiber tail VYPF was also
found in the fiber tail of OAV287 (Vrati et al., 1995) and EDSV (Hess et al., 1997).
Moreover, based on a conserved TLWT sequence it was possible to define where the
knob region of the fiber begins (Xia et al., 1994). Although, such a motif is conserved in
many adenoviruses, it is missing in the FAdV-8 fiber. Absence of the motif has been also
reported for OAV287 and EDSV (Vrati et al., 1995; Hess et al., 1997). Lastly, in most
adenoviruses including HAdV-5 (Chroboczek et al., 1992), canine (CAdV-1) (Morrison
et al., 1997), bovine (BAdV-3) (Mittal et al., 1992), porcine (PAdV-3) (Reddy et al.,
1995) and murine (MAdV-1) (Beard et al., 1990) adenoviruses, the E3 region is found
between the pVIII and fiber protein genes. E3 proteins are involved in the inhibition of
presentation of antigenic protein via the major class I histocompatibility complex (MHC-
I). The E3 genes of HAdVs are involved in the modulation of the host’s immune response
including cytotoxic T lymphocyte (CTL)-induced apoptosis (Wold and Gooding, 1991).
The E3 region represents one of the most commonly used insertion sites in the adenovirus
gene delivery system (Russell, 2000). However, deletion of this region results in an
increased inflammatory response when compared to the wild type virus counterpart
(Braithwaite and Russell, 2001). The size of the E3 region varies considerably among the
62
mastadenoviruses and the proteins encoded by this region are also different. Lack of an
E3 region has been reported in ovine (OAV287; Vrati et al., 1995) and several avian
adenoviruses, such as EDSV (Hess et al., 1997), FAdV-1 and FAdV-9 (Chiocca et al.,
1996; Ojkic and Nagy, 2000).
Our data demonstrated that genes in the central region of the genome were in the same
order as in every adenovirus studied to date. These include the genes of structural
proteins and the viral protease gene coded on the r strand.
In an investigation of any disease outbreak, commonly asked questions are about the
pattern of infection and how transmission occurs. From the available evidence it seems
that most of the strains of FAdVs follow the same pattern of infection. Following initial
multiplication there is probably viraemia that results in virus spread to virtually all
organs. The main sites of virus replication appear to be respiratory and alimentary tracts
(Saifuddin and Wilks, 1991). In our study the organ with the highest viral load was cecal
tonsil in both orally and im inoculated chickens. Ojkic and Nagy (2003) demonstrated
that replication and distribution in chicken tissues can be markedly influenced by route of
virus inoculation. However, horizontal transmission of FAdVs occurs through contact
with nasal and tracheal excretions and feces, the latter of which contains the highest virus
titers (Grgic et al., 2006). Therefore, we compared im and the more natural oral routes of
inoculation and the results showed that the im inoculated group had higher concentrations
of viral copies in general. Furthermore, virus was isolated from cloacal swabs up to 28 d.
p.i. and that was probably the longest recorded shedding period for fowl adenoviruses to
63
date (Corredor and Nagy, 2010a). The highest virus titers in the feces for the orally and
im-inoculated birds were recorded at days 10 and 3 p.i., respectively. Virus shedding
could be indirectly correlated to the Ab response of chickens. Indeed the development of
neutralizing Abs coincides with cessation of virus excretion (Clemmer, 1972) as was
possibly the case in this study. The lower Ab response in orally-inoculated chickens, the
lower viral genome copy number in tissues in the oral group and the lower virus titer in
the feces of orally-inoculated chickens could be attributed to the oral route of inoculation
and the amount of virus that indeed infected chickens. It is important to note, that
although not the natural route of viral infection, intramuscular inoculation ensures that all
birds receive the same amount of virus. That might be the reason why im-inoculated
chickens exhibited virus recovery and Ab results with higher values.
Our original hypothesis was that this virus was pathogenic since it was isolated from a
flock with clinical signs of inclusion body hepatitis. Thus a complete genome sequence
was determined and a pathogenicity study was performed. However, based on the
pathogenicity data we concluded that under the conditions of this study this isolate was
not pathogenic.
In summary our results for FAdV-8 are concordant with findings reported for the other
genomes of fowl adenoviruses. Along with FAdV-1 and FAdV-9, FAdV-8 lacked
mastadenoviral E1, E3 and E4 region, and sequences for protein V and IX. Similar to
FAdV-9, FAdV-8 has only one fiber gene. We also found that with regard to sequence
composition and genome organization, FAdV-8 is closer to FAdV-9 than to FAdV-1.
64
Genetic distance between species E (FAdV-8) and D (FAdV-9) also indicates a closer
relationship between them and this observation is supported by the high identities among
their ORFs (Table 2.1). Our findings suggest that FAdV-1 of species Fowl adenovirus A
as the current type species is, despite its historical priority not representative of the genus
Aviadenovirus, and that FAdV-8 or FAdV-9 in species Fowl adenovirus E and Fowl
adenovirus D, respectively, would be more suitable for that designation. Despite lack of
clinical signs and pathological changes virus was found in tissues and cloacal swabs in
the chickens inoculated with FAdV-8. However, chickens inoculated im had higher
antibody titers than the orally inoculated ones. This information is important in devising
vaccination strategies against inclusion body hepatitis and for developing FAdV-8 as a
recombinant vector for gene delivery or for vaccine application, as we have already
described and discussed for FAdV-9 (Corredor and Nagy, 2010a,b).
Acknowledgements
This work was supported by the Natural Sciences and Engineering Research
Council of Canada (NSERC), the Poultry Industry Council (PIC) of Ontario and the
Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA). The authors thank
Mrs. Sheila Watson for her technical help, and the Isolation Unit personnel for their
professional animal care and assistance.
65
Table 2.1. Percent identities/similarities of selected FAdV-8 proteins from this study and their homologues.
Serotype/Species ACCESSION IVa2 DNA pol pTP 52/55kDa pIIIa pIII pVII pX pVI
FAdV-1/A NC001720 64.9/77.9 66.2/75.1 59.8/65.5 67.8/78.1 66.8/77 73.5/81.8 62.4/69.4 55.5/65.6 32.3/43.3
FAdV-9/D NC000899 87.3/91 79.5/84.9 74.3/78.3 82.5/88.1 90.8/95.2 86/89.8 68.8/72.9 77.5/85.8 38.7/44.3
FAdV-4/C ACR54094 _a _ _ _ _ _ _ _ _
FAdV-8/E AAC55302 _ _ _ _ _ _ _ _ _
FAdV- 8/E CFA40 AAF17339 _ _ _ _ _ _ _ _ _
FAdV-10/C AAB88670 _ _ _ _ _ _ _ _ _
HAdV-2/C J01917 30.8/44.8 34.8/48 29.2/42.9 23.6/37 23.6/36.8 40.5/51.4 17.5/22.5 _ 17.4/27.8
Serotype/Species ACCESSION Hexon Protease DBP 100K 33kDa pVIII Fiber/sb Fiber/lc
FAdV-1/A NC001720 81.2/88.5 76.1/86.6 58/70.1 50.9/61 5.6/13.1 68.5/76.2 29/42.7 17.4/25.1
FAdV-9/D NC000899 88.3/92.2 87.1/93.8 73.7/82.1 53.7/61.6 24.7/29.1 84.1/86.6 53.4/67.2 _
FAdV-4/C ACR54094 _ _ _ _ _ _ 39/54.4 _
FAdV-8/E AAC55302 _ _ _ _ _ _ 67.4/72.3 _
FAdV-8/E CFA40 AAF17339 _ _ _ _ _ _ 66.5/69.2 _
FAdV-10/C AAB88670 _ _ _ _ _ _ 36.7/52.5 _
HAdV-2/C J01917 46.8/59.7 39/62.9 26.5/42 26.4/37.6 14.6/23 24.9/37.5 19.2/30.2 _
a= data not available b= short fiber c= long fiber Percent identities and similarities were calculated using FASTA3 (EMBL-EBI).
66
Table 2.2. Numbers of positive tissues and viral genome copy numbers in tissues of chickens inoculated with FAdV-8.
days p.i.
Inoculation route
tissue oral Im
Liver 6.7x102* 4/4§ 2.9x103 4/4
3 Bursa 4.8x100 3/4 9.6x103 4/4
Cecal tonsil 2.6x103 4/4 4.4x103 4/4
Liver 8.6x102 4/4 3.3x104 4/4
5 Bursa 1.3x102 4/4 1.6x105 4/4
Cecal tonsil 7.5x103 4/4 1.5x105 4/4
Liver 2x104 4/4 1.9x102 4/4
7 Bursa 1.3x101 4/4 3.8x102 4/4
Cecal tonsil 6.5x104 4/4 5.5x105 4/4
Liver 1.4x100 4/4 1.2x102 4/4
14 Bursa 6.1x101 3/4 5.8x100 3/4
Cecal tonsil 3.5x102 4/4 5.7x102 4/4
Liver 3.4x103 4/4 4.5x102 4/4
21 Bursa 2.2x100 2/4 7.9x102 3/4
Cecal tonsil 2x103 4/4 1.7x103 4/4
Liver 1.4x101 4/4 2.3x101 4/4
28 Bursa 5.3x100 4/4 1.1x101 3/4
Cecal tonsil 2.9x102 4/4 2.8x102 4/4
* = viral genome copy number/10 µg of total tissue DNA was determined by qPCR § = number of positive tissues/number analyzed tissues
67
Table 2.3. Virus titers in the feces of chickens inoculated with FAdV-8.
Days Inoculation route post-infection Titers (pfu/ml) in swabs
Chickens shedding the virus (%)†
Oral Im 9.4x103±1.7x103 3.1x104±7x103
3 P=0.33 (100) (100) 1.3x104±4.2x103 1.9x104±4.4x103
5 P=0.32 (96) (100)
1.6x104±8x103 9.6x102±3x102
10 P<0.01 (96) (25)
7.9x102±3x102 1x102±3x101
14 P=0.18 (86) (31)
0.8x101±0.8x101 2.9x102±1x101
21 P<0.006 (8) (58)
2.2x101±1x101 3.2x102±1.7x101
28 P<0.003 (22) (85)
†30 chickens at day 0; 26 and 22 chickens at days 3 and 5, respectively; 18
and 14 chickens at days 10 and 14, respectively; 10 and 6 chickens at days 21 and 28, respectively. The virus titers were determined by plaque assay.
P values < 0.05 are statistically significant.
68
Supplementary Table 2.1. Position of 46 predicted genes potentially encoded by the genome of FAdV-8. Gene Strand Location No. of aa G+C content (%) ORF0 r 515-799 95 61 ORF1 r 837-1298 154 61 ORF1A/B r 1493-1705 71 56 ORF1C r 1605-1871 89 55 ORF2 r 1922-2728 269 58 ORF24 l 3470-2901 190 56 ORF14 r 3947-3600 116 60 ORF13 l 4620-4315 102 58 Unknowna r 4891-5328 146 60 ORF12 l 6010-5255 252 55 IVa2 l 7350-6208 381 57 DNA pol l 11027-7347 1227 63 pTP l 13478-11259 740 63 52/55kDa r 13655-14884 410 64 pIIIa r 15017-16636 540 64 pIII r 16796-18454 553 61 pVII r 18492-18728 79 63 pX r 18952-19542 197 65 pVI r 19718-20416 233 66 Hexon r 20531-23374 948 57 Protease r 23405-24034 210 61 DBP l 25542-24142 467 63 100K r 26164-29262 1033 60 33kDa r 28910-29338 143 62 pVIII r 29885-30610 242 63 U-exon l 30864-30565 100 54 Fiber r 30863-32424 524 55 ORF32 l 31431-31204 76 57 ORF22a l 32950-32501 150 59 ORF22b r 33080-32916 55 59 ORF20A l 33638-33084 185 57 ORF20 r 34457-33648 270 53 ORF19 l 36807-34726 694 49 ORF28 r 37926-38243 106 42 ORF29 r 38470-38670 67 59 ORF8a r 38652-39047 132 58 ORF8b r 39052-39486 145 60 ORF8c r 39291-39551 87 61 ORF17 l 40152-39682 157 53 ORF33a r 40106-40300 65 58 ORF33b r 40502-40666 55 58 ORF11 r 40513-41202 230 55 ORF11A l 41553-41344 70 47 ORF23 l 42715-41852 288 55 ORF25a r 43147-43773 209 57 ORF25b r 43248-43706 153 59
The size of the deduced proteins and the G+C content of the individual genes are also shown. a Novel unidentified open reading frame r = rightward transcribed strand l = left transcribed strand.
69
Figure 2.1. Schematic representation of FAdV-8, FAdV-9 and FAdV-1 genomes, members of the genus Aviadenovirus. Pink arrows depict genus common genes, blue arrows show genes conserved in all adenovirus genera, green arrows represent unique genes in FAdV-8 and FAdV-1 genome, red arrows depict genus specific genes and the yellow arrow shows unidentified ORF.
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Figure 2.2. Antibody response to viral proteins in chickens inoculated with FAdV-8 by the oral and intramuscular routes and mock infected as measured by S/P ratios. Serum samples were collected at 7, 14, 21 and 28 days post-infection. Dark gray and white columns represent im and orally inoculated chickens, respectively. Light gray and black columns represent mock-infected chickens. The error bars correspond to 95% confidence intervals. Significant differences among mock infected and infected birds are indicated by asterisks (*).
71
CHAPTER 3 Key cytokines activated during infection of chickens with a serotype 8 fowl
adenovirus
Helena Grgića, Shayan Sharifa, Hamid R. Haghighia and Éva Nagya,*
aDepartment of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, N1G 2W1, Canada
*Corresponding author: Éva Nagy E-mail: [email protected] Tel: +1 519 824 4120 ext 54783 FAX: +1 519 824 5930
Author’s contributions
HG designed and performed experiment, carried out analysis and wrote the paper. HRH
provided guidance for real-time PCR. SS and EN provided guidance during the research
and critically revised manuscript
Submitted to Virus Research
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Abstract
This study explored the ability of fowl adenovirus (FAdV) to subvert the host
cells expression of cytokines in response to infection as an important viral mechanism for
immune evasion during infection. The production of certain cytokine mRNA was
quantified by real-time quantitative reverse transcription-PCR (RT-PCR) in spleen, liver
and cecal tonsil during the course of infection of chickens with a serotype 8 FAdV
(FAdV-8). Compared to uninfected chickens, infected birds had higher mRNA
expression of interleukin (IL) 18 and IL-10 in spleen and liver, respectively. Interferon
gamma (IFN-γ) mRNA expressed in spleen and liver of infected chickens was
significantly up-regulated, while the expression of IL-8 mRNA in spleen and liver of
infected chickens was significantly down-regulated. There was no significant difference
between infected and uninfected groups in terms of cytokine gene expression in cecal
tonsil. These results indicate that these four cytokines might play a crucial role in driving
the immune responses following FAdV-8 infection.
Keywords:
Fowl adenovirus serotype 8; interleukins; IFN-γ; IL-18; IL-10; IL-8.
Abbreviations: FAdV-8, fowl adenovirus 8; d.p.i., days post-infection; pfu, plaque
forming unit; SPF, specific pathogen free; IFN-γ, interferon gamma; IL, interleukin;
h.p.i., hours post-infection; IBH, inclusion body hepatitis; MOI, multiplicity of infection;
IRF3, interferon regulatory factor; CAR, coxsackie and adenovirus receptor; PAMs,
pathogen-associated molecular patterns; TLR, toll like receptors; NK, natural killer cells;
DCs, dendritic cells.
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Highlights:
First study of cytokine gene expression following fowl adenovirus infection of chickens
FAdV-8 infection is associated with up-regulation of IL-18 and IFN-γ mRNA expression
Up-regulation of IL-10 is indicative of an immune evasive mechanism used by FAdV-8
FAdV-8 infection suppresses IL-8 gene expression in chickens
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1. Introduction
Fowl adenoviruses (FAdVs) are in the family Adenoviridae, genus Aviadenovirus
and are classified into five species, Fowl adenovirus A-E, based on their DNA genomes
and further divided into 12 serotypes based on virus neutralization (Benkő et al., 2005).
FAdVs have a worldwide distribution and some are associated with diseases such
as inclusion body hepatitis (IBH). IBH is mainly seen in meat type chickens 3-7 weeks of
age (Adair and Fitzgerald, 2008). However, some FAdVs cause no or mild clinical signs
and could be isolated from asymptomatic chickens, and are considered ubiquitous in
poultry populations. Clinical signs of IBH are characterized by sudden onset of mortality
that usually ranges from 2-10% in affected flocks. Mortality varies and is closely related
to the pathogenicity of the virus, secondary infection with other pathogens and
susceptibility of the chickens. At gross examination the main lesions are seen in the liver
which is swollen, light brown to yellow, occasionally with hemorrhages and necrotizing
hepatitis. Histologically the basophilic nuclear inclusion bodies present in the hepatocytes
are characteristic of infection (Philippe et al., 2005). Most of the FAdV strains follow the
same pattern of infection, after initial multiplication the virus spreads to virtually all
organs. Viraemia is detected at 24 hours post infection (h.p.i.) and has two peaks, the first
at 2 and the second at 7 day post infection (d.p.i.) (Saifuddin and Wilks, 1991). FAdVs
are transmitted both vertically and horizontally, and the virus is present in all excretions
such as those from the feces, trachea and nasal mucosa. The usual virus transmission
within a flock is via the oral-fecal route (Cook, 1974; Adair and Fitzgerald, 2008; Grgić
et al., 2006).
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Members of the genus Aviadenovirus have the largest genome (43-45 kb) among
adenoviruses, despite the fact that a few structural protein genes such as V and IX, and
early regions E1, E3 and E4 described in mastadenoviruses are not identified for
aviadenoviruses (Chiocca et al., 1996; Ojkic and Nagy, 2000; Kajan et al., 2010; Griffin
and Nagy, 2011; Grgić et al., 2011).
The early gene products of mammalian adenoviruses are responsible for host cell
interactions and modulation of the immune response including cytokine expression by
encoding proteins, which counteracts TNF-α (Lesokhin et al., 2002) or the virus-encoded
RNA (VA RNA), which prevents interferon-mediated shut-off of protein synthesis (Wold
et al., 1994) among others.
Earlier we determined the pathogenicity of a fowl adenovirus serotype 8 (FAdV-
8) isolate based on clinical signs, pathological and histological examination.
Additionally, viral genome copy numbers in the bursa of Fabricius, cecal tonsil and liver
following infection were evaluated (Grgic et al., 2011). Moreover, virus titer in feces and
antibody response were also determined. Despite a lack of clinical signs and pathological
changes virus was found in all selected tissues and cloacal swabs of all birds. The current
work is a continuation of our efforts towards better understanding the pathogenesis of
FAdV infection with the emphasis on the role of interleukins, which is an unknown area.
Our objectives were to investigate the effects of FAdV infection on the
transcription of a number of avian cytokines, both in vitro and in vivo. First, the dynamics
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of cytokine gene expression in FAdV-8 infected chicken hepatoma cells (CH-SAH) at
different time points was determined. Then in a chicken experiment interferon gamma
(IFN-γ), interleukin (IL) 10, IL-18 and IL-8 gene expression was followed in spleen, liver
and cecal tonsil at different days after infection with FAdV-8, and compared to that of
uninfected chicken samples by quantitative real-time RT-PCR.
2. Materials and methods 2.1. Virus and cells
FAdV-8 was isolated by the Animal Health Laboratory (AHL) at the University
of Guelph and characterized in our laboratory (Grgić et al., 2011). Virus propagation and
titration were performed in CH-SAH cells as described by Alexander et al. (1998).
CH-SAH cells were maintained in Dulbecco’s Modified Eagle’s Medium/Nutrient
Mixture F-12 Ham (DMEM-F12) with 10% non-heat inactivated fetal bovine serum
(FBS) as described (Alexander et al., 1998). To determine the effect of FAdV-8 on the
induction of cytokines, CH-SAH cells were plated onto 35-mm-diameter Petri dishes and
infected with a multiplicity of infection (MOI) of 10. At 1, 4, 8, 10, 12 and 24 hours post-
infection (h.p.i.), the cells were harvested and processed for RNA extraction. Total RNA
was prepared from triplicate cultures at each time point using TriZol (Invitrogen, Canada
Inc., Burlington, ON, Canada) as per the manufacturer’s protocol.
2.2. Experimental design
Specific pathogen free (SPF) eggs were obtained from the Canadian Food
Inspection Agency (CFIA) (Ottawa, Ontario, Canada), hatched at the Arkell Poultry
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Station, University of Guelph and the chicks were housed at the University Isolation
Unit. Ten-day old chicks were randomly divided into two groups, each group containing
50 birds. Group I was assigned for intramuscular (im) administration of the virus, each
bird received 2x108 plaque forming units (pfu). Group II, the uninfected chicks (negative
control) received PBS by im administration. Following inoculation, five chicks from each
group were randomly drawn for necropsy at 1, 2, 3, 4, 5, 6, 7, 10, and 14 d.p.i. Birds were
assigned for necropsy by simple random sampling of their identification numbers
stratified by cage, using the statistic software (MINITAB 14). Euthanasia was performed
according to the University of Guelph Animal Care Committee regulations (Isolation
Unit, University of Guelph, Ontario, Canada) and the spleen, cecal tonsil and liver were
collected, and immediately submerged in RNA stabilization reagent (RNAlater, Qiagen,
Mississauga, ON, Canada) and stored at -80°C until RNA extraction.
2.3. RNA extraction
Total RNA was extracted from the tissue samples by TriZol reagent (Invitrogen,
Canada Inc., Burlington, ON, Canada) as described by the manufacturer. Briefly, tissues
were homogenized in 1 ml TriZol followed by chloroform extraction, isopropanol
precipitation and a 75% ethanol wash of the RNA. The pellet was dissolved in 20 µl of
RNAse free water and the RNA concentration was measured by NanoVue
spectrophotometry. To remove possible contaminating DNA the samples were treated
with a DNase based inactivation reagent (Invitrogen, Canada Inc., Burlington, ON,
Canada). The cDNA was synthesized with Random Primer (Invitrogen, Canada Inc.,
Burlington, ON, Canada) and by using SuperScriptTM II Reverse Trancriptase
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(Invitrogen, Canada Inc., Burlington, ON, Canada) according to the manufacturer’s
instructions. The cDNAs were stored at -80°C until analyzed by real-time PCR.
2.4. Preparation of constructs for producing standard curves
Preparation of constructs and creation of standard curves for all cytokine genes,
which were used in this study, as well as the β-actin gene were as described (Abdul-
Careem et al., 2006; 2007).
2.5. Real-time PCR
Real-time PCR was used to quantify the expression of β-actin and cytokine genes
from the cDNA samples. The PCR reaction was prepared in a total volume of 20 µl of
Qiagen Quantitect SYBR Green PCR kit (Qiagen) containing hot start Taq polymerase
and cDNA. All the real-time PCR reactions were carried out in 96-well plates (Roche
Diagnostics GmbH, Mannheim, Germany). Each assay was run along with no-template
control and three dilution series from the standard. The sequences and other features of
specific primers for chicken IFN-γ, IL-18, IL-10, IL-8, and β-actin have been described
(Abdul-Careem et al., 2006; 2007). The primers were synthesized by Laboratory
Services, University of Guelph. The PCR conditions for different segments of each cycle
were optimized for all target genes because the thermal cycling parameters differ for each
gene (Abdul-Careem et al., 2006). All samples were amplified in a LightCycler (LC480)
instrument (Roche Diagnostics GmbH, Mannheim, Germany).
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2.6. Data analysis
Real-time PCR efficiency was calculated by 10(-1/slope of the standard curve) (Rasmussen,
2001). The mRNA expression was calculated relative to β-actin gene expression using
the equation (Etarget)ΔCPtarget(control –sample)/(Eref)
ΔCPref(control –sample) (Pfaffl, 2001). In this
equation, CP (crossing point) is the point at which the fluorescence rises significantly
above the background. The difference in cytokine expression between groups was
assessed by Mann-Whitney test and comparisons were considered significant at P≤0.05.
3. Results 3.1. Cytokine gene expression in CH-SAH cells
In our laboratory FAdVs are propagated in CH-SAH cells and since there are no
data available about the expression of cytokines by these cells we followed IFN-γ, IL-18,
IL-10 and IL-8 mRNA production upon infection with FAdV-8. The expression of IFN-γ,
IL-18, IL-10 and IL-8 of uninfected and infected CH-SAH cells at various time points is
illustrated in Fig. 3.1. The gene expression of all selected cytokines was down-regulated
in the first four hours after infection when compared to uninfected controls. The
expression of IL-10, IL-18 and IL-8 was up-regulated at 8 h.p.i. and the difference
between infected and uninfected samples was statistically significant (P≤0.05).
Significant up-regulation was detected up to 10 h.p.i. for IL-10 and IL-8 while for IL-18
up-regulation persisted up to 12 h.p.i. IFN-γ exhibited significant (P≤0.05) transient up-
regulation measured only at 10 h.p.i.
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3.2. Cytokine gene expression in experimentally infected chickens
The cytokine gene expression in the spleen, liver and cecal tonsil of chickens
infected with FAdV-8 was evaluated by a real-time quantitative reverse transcription-
PCR.
3.2.1. Gene expression in spleen
The expression of IFN-γ, IL-18, IL-10 and IL-8 mRNA in spleen of FAdV-8
infected and uninfected control chickens is shown in Fig. 3.2. The expression of selected
interleukins was detected at all time points. There was a statistically significant increase
(P≤0.05) in IFN-γ gene expression in spleen of infected chickens on day 3 p.i. Also, at
this time point, there was a significant (P≤0.05) decrease in IL-10 gene expression.
However, when IL-18 gene expression was compared to that of uninfected controls
statistically significant (P≤0.05) increase in expression was seen among infected chickens
at day 1 p.i. Expression of this cytokine changed on day 7 p.i., when it was significantly
down-regulated (P≤0.05) in the infected group. IL-8 gene expression showed similar
patterns to those of other cytokines in that it was expressed across all time points in the
spleen of chickens belonging to both infected and uninfected groups. Expression of this
cytokine was significantly down-regulated (P≤0.05) in the infected group at days 7 and
10 p.i.
3.2.2. Gene expression in cecal tonsil
There were no significant differences (P>0.05) in expression of mRNA for any of
the tested cytokine genes, compared with the values for the appropriate uninfected
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controls (Fig. 3.3). Increased levels of IFN-γ, IL-18 and IL-10 mRNA were evident in
infected birds at 1 d.p.i. although the results were not statistically significant. Patterns of
mRNA expression changed after 1 d.p.i. for IFN-γ, IL-18, IL-10 showing a downward
trend over the course of the experiment. IL-8 gene expression was down-regulated and
although the expression of IL-8 was detected at all time points in both groups, there was
no significant difference in the expression between groups (Fig. 3.3).
3.2.3. Gene expression in liver
The expression of IFN-γ, IL-18, IL-10 and IL-8 mRNA in liver of infected and
control chickens are illustrated in Fig. 3.4. Marked differences in expression of mRNA
between infected and uninfected chickens were evident for all studied cytokines. There
was a statistically significant increase (P≤0.05) in IFN-γ gene expression in liver of
infected chickens on days 3, 5, and 7 p.i. Infected chickens also had a significantly higher
IL-10 gene expression compared to chickens in the uninfected control group at days 1, 3,
and 5 p.i. Additionally, adenoviral infection altered the expression levels of IL-8 and IL-
18 and resulted in an increased expression (P≤0.05) of IL-18 at 3 d.p.i. On the other hand
the expression of IL-8 in the liver was down regulated and a decrease in expression
became more prominent and statistically significant (P≤0.05) at days 5, 7, and 10 p.i.
4. Discussion
Fowl adenoviruses are widely distributed in poultry operations and infect a wide
range of tissues, but their ability to cause disease depends on numerous factors such as
the type and age of the chickens, virulence of the virus, and secondary infection. The host
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defense mechanism in healthy birds usually functions effectively, however, some isolates
of certain serotypes can cause considerable morbidity and mortality especially among
nutritionally or immunologically compromised birds caused by a different viral infection,
for example chicken anemia or infectious bursal disease viruses (Toro et al., 2000). Due
to an increased interest in adenoviruses as vaccine and gene delivery vectors it is
important to understand the molecular aspects of infection and the host responses to them
(Corredor and Nagy, 2010b).
Innate early response occurs as a direct consequence of interaction of the virus
with the cell and does not always depend on the transcription of any virus genes.
Nevertheless, the nature of the innate response can differ significantly depending on
receptor usage and cell type, activating a complex signaling cascade with diverse
outcomes. Rendall and Goodbourn (2008) showed that the majority of these pathways
lead to the up-regulation of transcription factor NF-κB and interferon regulatory factor 3
(IRF3). Muruve (2004) confirmed that the interaction between adenoviral capsid and host
cell is sufficient to activate the pathways that lead to inflammatory responses. A study of
Tamanini et al. (2006) demonstrated that the fiber is sufficient for the immediate pro-
inflammatory response. The interaction of the fiber with the Coxsackie and adenovirus
receptor (CAR) is a critical initial event for such a response. Following adenovirus
infection, pathogen-associated molecular patterns (PAMs) are recognized by cellular
sensors, pattern-recognition receptors (PRRs) (Akira et al., 2006). The main function of
PRRs comprise enhancement of phagocytosis, activation of complement cascades,
production of inflammatory cytokines and maturation of dendritic cells (DCs), that all
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together orchestrate the early host response to infection and are important link to the
adaptive immune response (Akira et al., 2006). The most extensively studied PRRs are
toll-like receptors (TLR). To date 13 TLRs (TLR1 to TLR13) have been identified in
mammals and mice. In chickens Temperley et al. (2008) found nine TLRs (1, 2, 3, 4, 5, 7,
9, 15, and 21) with five human and one fish orthologs, and only three are unique to
chicken (Fukui et al., 2001; Kogut et al., 2005; Boyd et al., 2007; Jenkins et al., 2009).
Data on the interaction of FAdV-encoded molecular patterns and TLRs that recognize
these PAMPs are not available. However, there are differences in innate immune
response pathways depending on the infected cells and on the adenovirus species. Kim et
al. (2008) reported the recognition of unmethylated CpG sequences in human adenovirus
by TLR9. There are essential differences in the TLR repertoires of mammals and birds,
TLR15 does not exist in mammals and chickens lack TLR9. Keestra et al. (2010)
reported chicken TLR21 as a CpG DNA receptor that shares functional characteristics but
displays minimal sequence similarity with mammalian TLR9. Activation of innate
immune response without the participation of TLR9 has been reported by Takaoka et al.
(2007). Cerullo et al. (2007) showed that empty adenoviral particles devoid of genomic
DNA are poor inducers of innate responses suggesting that the DNA is a major
immunostimulatory molecule of adenoviruses. Engagement of some of the innate
receptors, such as TLRs with PAMPs, results in triggering of downstream pathways,
including IFN. Another type I or Th1 cytokine, IL-18 is critical for IFN- γ production by
natural killer (NK) cells and T cells (Rathinam and Fitzgerald, 2010). On the other hand,
the potent chemotactic cytokine IL-8 is known as an important mediator of inflammation
which recruits and activates neutrophils to sites of infection (Baggiolini, 1998). The
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overproduction of cellular IL-8 in viral infections is a reactive response of the host cells
aimed to reducing viral load (Parcells et al., 2001). On other hand, IL-10 has a key effect
on the suppression of Th1 cell response. This cytokine inhibits the production of the pro-
inflammatory cytokines by DCs and macrophages (Moore et al., 2001). Additionally, IL-
10 modulates the immune response towards a type 2 response by inhibiting IFN-γ
synthesis (Rothwell et al., 2004).
We examined cytokine gene expression patterns associated with FAdV infection
and to our knowledge this is the first report on this subject. We determined that IFN-γ,
IL-10, IL-18 and IL-8 were activated at early times after FAdV-8 infection. The studied
cytokines represent chicken Th1 and regulatory cytokines and all have previously been
shown to be involved in immunity to viral infections.
IL-18 is an important regulator of innate and acquired immune response and is
expressed at sites of inflammation (Gobel et al., 2003). Although originally identified as
an inducer of IFN-γ production, this cytokine also enhances T and NK cell maturation,
cytokine production and cytotoxicity (Okamura et al., 1995; Yoshimoto et al., 1998;
Micallef et al., 1996; Dao et al., 1998). During viral infection IL-18 activates CD8+ T
cells and is important for viral clearance. Abdul-Careem et al. (2007) showed that IL-18
expression is significantly higher in spleen of chickens in the unvaccinated/infected
group in which IL-10 expression was enhanced, whereas IFN-γ expression was minimal.
A possible explanation for such result was that chicken IL-18 could also be involved in
inducing type 2 cytokines, such as IL-10.
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Similar to mammals (Endharti et al., 2005; Minang et al., 2006) one of the roles of
chicken IL-10 is to repress the Th1 response. IL-10 aborts T cell responses and can
inhibit ongoing T cell activity to viral infections. It has been shown by several groups
(Carbonneil et al., 2004; Fiorentino et al., 1991; Moree et al., 2001; Steinbrink et al.,
1997) that IL-10 alters cytokine production and prevents maturation depressing T cell
activation.
In agreement with the findings of Abdul-Careem et al. (2007) we discovered that
expression of IL-18 and IL-10 genes in infected CH-SAH cells was significantly up-
regulated at 8, 10 and 12 h.p.i. in comparison with mock infected cells. Hence, IFN-γ
expression was significantly up-regulated at a single time point (10 h.p.i) and shortly
after exhibited a tendency to down-regulation. Our potential explanation of such a shift
in expression of IFN-γ is the inhibitory effect of IL-10 as observed by Endharti et al.
(2005) and Minang et al. (2006).
A slightly different trend was seen in chickens where expression of IL-18 gene
was significantly higher in spleen and liver of infected chickens at day 1 and 3 p.i.,
respectively. Accordingly, IL-10 expression was significantly up-regulated in liver of
infected chickens at 1, 3, and 5 d. p .i. In our study a significant induction of IFN-γ gene
was noted in the spleen of infected chickens at day 3 p.i., while in the liver the expression
of IFN-γ was significantly higher at day 3, 5 and 7 p.i. The reported variation might be
directly related to the pathogen used in the study. Moreover, although IL-10 is considered
a regulatory cytokine, which is inversely correlated with the expression of Th1 cytokines
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such as IFN-γ, under certain circumstances could be produced by Th1 cells (O’Garra and
Vieira, 2007). Further, IL-12 stimulates human CD4 and CD8 T cells for increased
production of both IFN-γ and IL-10 (Gerosa et al., 1996). Simultaneous expression of
IFN-γ and IL-10 genes has been also reported by Johensen et al. (2004) following
infection with porcine reproductive and respiratory syndrome virus.
Adenovirus encodes numerous products that counteract host defenses, ensure
prolonged survival in the infected host and consequently facilitate viral transmission.
Zhou et al. (2007) have demonstrated that porcine adenovirus 3 early region 1Blarge
(E1Blarge) protein down-regulates the induction of proinflammatory cytokine IL-8 by
inhibiting the NF-κB dependent gene transcription from human IL-8 promoter. The same
authors suggested that the E1Blarge protein acts as a IkappaB (IκB) homolog and retains
the ability to bind and inactivate NF-κB. Moreover, Lesokhin et al. (2002) reported that
the E3 genes can drastically reduce level of IL-8 mRNA. Inhibition of the amounts of
chemokine mRNA by E3 is directly related to decreased secretion of IL-8 proteins as
showed by ELISA. Four out of six proteins of the human adenovirus E3 region are
involved in immunoregulation (Wold et al., 1994). Bennett et al. (1999) showed that E3-
19K retains the major histocompatibility complex (MHC) I in the endoplasmic reticulum
and inhibits tapasin processing of peptides that bind to MHC I. The E3-10.4K and E3-
14.5K complex stimulates the internalization of proapoptotic receptors from the cell
surface (Tollefson et al., 1998; Elsing and Burgert 1998). The E3-14.7 inhibits tumor
necrosis factor alpha (TNF-α)-induced apoptosis (Gooding et al., 1990; Li et al., 1999).
Additionally, E3-14.7K inhibits TNF-α-induced production of arachidonic acid (Krajcsi
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et al., 1996). It is unclear which E3 protein interactions are important in the inhibition of
IL-8. The E3 region is placed between the pVIII and fiber protein genes and deletion of
this region results in an increased inflammatory response (Braithwaite and Russell 2001).
We recently showed that FAdV-8 along with FAdV-1, FAdV-4 and FAdV-9 lacks
mastadenoviral E1, E3 and E4 regions (Grgic et al., 2011).
In CH-SAH cells expression of IL-8 in infected cells was significantly down-
regulated in comparison with mock infected cell at 1, 4, and 24 h.p.i. A similar
observation has been reported in Marek’s disease virus (MDV)-transformed
lymphoblastoid cell line where infection with Mycoplasma gallisepticum suppressed IL-8
gene expression (Lam, 2004). On other hand, IL-8 is up-regulated in splenocytes after
Marek’s disease virus infection (Xing and Schat, 2000).
In this study, the expression of IL-8 mRNA in spleen of infected chickens was
significantly down-regulated at 7 and 10 d.p.i., similarly, significant down-regulation was
detected in the liver at 5, 7 and 10 p.i. These results are in accordance with the findings of
Zhou et al. (2007) and Lesokhin et al. (2002). Interestingly, the GAM-1 protein, encoded
by the Gam 1 gene (ORF8) at the right end of the FAdV-1 genome, has mastadenoviral
E1 functions such as inhibition of apoptosis (Chiocca et al., 1996; Lehrmann and Cotten,
1999). Homologue of ORF8 has been also identified for FAdV-8 genome (Grgic et al.,
2011). Therefore, we speculate that some parts of the FAdV-8 genome share functions
similar to mastadenoviral E1, E3 and E4 regions despite a lack of obvious and high
sequence homology.
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In conclusion the results presented here showed that FAdV-8 infection is
associated with enhanced expression of IFN-γ, IL-18 and IL-10 and reduced expression
of IL-8 in spleen and liver of infected chickens. There was no significant difference in
any of the studied cytokine gene expression between infected and uninfected groups in
cecal tonsil. Our finding that IL-10 was significantly up-regulated in liver might be
indicative of an immune evasive mechanism used by the virus to skew the Th-1-like
immune response against FAdV-8 and to aid the persistence of the virus. Another
possible explanation could be that IL-10 was induced by the chicken immune response
and inflammation caused by the virus. Further work is necessary to shed more light on
the role of cytokines in elicitation of the immune response to FAdV infections.
Acknowledgements
This work was supported by the Natural Sciences and Engineering Research Council of
Canada (NSERC), the Canadian Poultry Research Council (CPRC), and the Ontario
Ministry of Agriculture, Food and Rural Affairs (OMAFRA). The authors thank the
Isolation Unit personnel for their professional animal care and assistance.
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Figure 3.1. Cytokine mRNA expression in CH-SAH following FAdV-8 infection. The groups were as follows: Uninfected (negative control) CH-SAH, and FAdV-8 infected CH-SAH. Target and reference gene expression was quantified by real-time RT-PCR using SYBR Green. Target gene expression is presented relative to β-actin expression and normalized to a calibrator. The results are diagrammed as whisker boxes with medians. The difference in cytokine expression between groups was assessed by Mann-Whitney test and comparisons were considered significant at P≤0.05 (*). Numbers 1, 4, 8, 10, 12, and 24 represent hours post infection when samples were collected.
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Figure 3.2. Cytokine mRNA expression in spleen of chickens infected with FAdV-8. The groups were as follows: Uninfected (negative control) chickens, and FAdV-8 infected chickens. Target and reference gene expression in spleen was quantified by real-time RT-PCR using SYBR Green. Target gene expression is presented relative to β-actin expression and normalized to a calibrator. The results are diagrammed as whisker boxes with medians. Boxes represent interquartile ranges and whiskers indicate extreme values. The difference in cytokine expression between groups was assessed by Mann-Whitney test and comparisons were considered significant at P≤0.05 (*). ○, ● symbols represent values which were identified as outliers. Numbers 1, 3, 5, 7, and 10 represent days post infection when samples were collected.
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Figure 3.3. Cytokine mRNA expression in cecal tonsil of chickens infected with FAdV-8. The groups were as follows: Uninfected (negative control) chickens, and FAdV-8 infected chickens. Target and reference gene expression in spleen was quantified by real-time RT-PCR using SYBR Green. Target gene expression is presented relative to β-actin expression and normalized to a calibrator. The results are diagrammed as whisker boxes with medians. Boxes represent interquartile ranges and whiskers indicate extreme values. The difference in cytokine expression between groups was assessed by Mann-Whitney test and comparisons were considered significant at P≤0.05 (*). ○, ● symbols represent values which were identified as outliers. Numbers 1, 3, 5, 7, and 10 represent days post infection when samples were collected.
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Figure 3.4. Cytokine mRNA expression in liver of chickens infected with FAdV-8. The groups were as follows: Uninfected (negative control) chickens, and FAdV-8 infected chickens. Target and reference gene expression in spleen was quantified by real-time RT-PCR using SYBR Green. Target gene expression is presented relative to β-actin expression and normalized to a calibrator. The results are diagrammed as whisker boxes with medians. Boxes represent interquartile ranges and whiskers indicate extreme values. The difference in cytokine expression between groups was assessed by Mann-Whitney test and comparisons were considered significant at P≤0.05 (*). ○, ● symbols represent values which were identified as outliers. Numbers 1, 3, 5, 7, and 10 represent days post infection when samples were collected.
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CHAPTER 4
Pathogenicity and cytokine gene expression patterns of a serotype 4 fowl adenovirus isolate, a vaccine candidate
Helena Grgića, Zvonimir Poljakb, Shayan Sharifa and Éva Nagya,*
aDepartment of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, N1G 2W1, Canada bDepartment of Population Medicine, Ontario Veterinary College, University of Guelph, Guelph, Ontario, N1G 2W1, Canada
*Corresponding author: Éva Nagy E-mail: [email protected] Tel: +1 519 824 4120 ext 54783 FAX: +1 519 824 5930
Author’s contributions
HG designed and performed experiment, carried out analysis and wrote the paper. ZP
advised on statistical analysis. SS and EN provided guidance during the research and
critically revised manuscript
To be submitted to Vaccine.
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Abstract
Infectious hydropericardium-hepatitis syndrome (HHS) is a recently emerged
disease of poultry associated with some strains of fowl adenovirus serotype 4 (FAdV-4).
In this study, a Canadian FAdV-4 isolate was evaluated for pathogenicity after oral and
intramuscular (im) infection of specific pathogen free (SPF) chickens. Pathogenicity was
evaluated by observation of clinical signs and gross and histological lesions. The highest
viral copy numbers, irrespective of the inoculation route, were detected in the cecal
tonsils. Virus titers in cloacal swabs collected over the entire study period were compared
between the orally and im inoculated chickens, and the difference in titers between the
two groups was significant (P<0.001). The antibody response of infected chickens tested
by an adenovirus-specific ELISA showed a statistically significant (P<0.001) difference
between the orally and im inoculated chickens. In addition, the effects of FAdV-4
infection on transcription of a number of avian cytokines were studied in vivo. Interferon
(IFN)-γ, interleukin (IL)-10, IL-18, and IL-8 were activated at early times after FAdV-4
infection. Our study demonstrated that this FAdV-4 isolate under experimental conditions
was non-pathogenic and could be considered as a live vaccine against HHS.
Keywords:
Fowl adenovirus serotype 4 (FAdV-4); Pathogenesis; IFN-γ; IL-18; IL-10; IL-8
Abbreviations: FAdV-4, fowl adenovirus 4; d.p.i., days post-infection; pfu, plaque
forming unit; SPF, specific pathogen free; IFN-γ, interferon gamma; IL, interleukin;
HHS, hydropericardium-hepatitis syndrome; MOI, multiplicity of infection; NK, natural
killer cells; Ab, antibody
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Highlights:
Pathogenicity of an FAdV-4 isolate in SPF chickens
Expression of cytokine genes after fowl adenovirus serotype 4 infection of chickens
FAdV-4 infection is associated with up-regulation of IL-18 and IFN-γ mRNA expression
Up-regulation of IL-10 may be indicative of an immune evasive mechanism used by
FAdV-4
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1. Introduction
Fowl adenovirus serotype 4 (FAdV-4) plays a major role in the aetiology of a
disease in broiler chickens called infectious hydropericardium-hepatitis syndrome (HHS).
This is an emerging disease initially reported from Pakistan (Khawaja et al., 1988). In
addition to India and Pakistan, outbreaks have also been reported in the Middle East,
Japan, Mexico, Peru, Ecuador, and Chile (Abe et al., 1998; Hess et al., 1999; Toro et al.,
1999). Outbreaks of HHS have not been reported in Canada. The disease is prevalent in
3- to 7-week-old broiler chickens. The affected birds may not exhibit signs other than
high mortality, up to 75% (Jaffery, 1988). The predominant gross lesion is
hydropericardium, characterized by accumulation of a jelly-like fluid in the pericardial
sac. The heart appears misshapen and flabby, with its apex floating in the pericardial sac
(Adair and Fitzgerald, 2008). At histopathological examination, the most consistent
findings in the liver are small multifocal areas of necrosis and mononuclear cell
infiltration, including basophilic intranuclear inclusion bodies in hepatocytes surrounded
by a clear halo or filling the entire nucleus (Nakamura et al., 1999). Initially, it was
hypothesized that HHS is caused by a toxin or by a nutritional disorder (Jaffery, 1988).
However, all attempts to reproduce the disease with feed samples from infected farms
failed. On the basis of successful isolation of an agent from the liver, heart, and kidneys,
and on transmission of disease after subcutaneous inoculation of infected liver
homogenate, the aetiological agent was tentatively identified as an adenovirus (Khawaja
et al., 1988). Subsequent studies demonstrated typical icosahedral adenovirus particles in
the purified liver extracts by negative-staining electron microscopy. The virus is usually
transmitted horizontally in flocks by the oral-fecal route (Cowen, 1992). Existing
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vaccines against HHS are composed of liver homogenate extract or virus propagated in
chicken liver cells or embryonated eggs. These preparations are inactivated with
formalin, β-propiolactone or heat treatment at 56˚C (Anjum, 1990) and they provide
protection against natural outbreaks of the disease (Anjum, 1990).
The FAdV-4 isolate investigated in this study was from a Canadian broiler
breeder flock with no clinical signs of HHS. The complete nucleotide sequence of the
virus was determined by Griffin and Nagy (2011).
The goals of this study were to (1) study the pathogenicity of this FAdV-4 isolate
in specific pathogen free (SPF) chickens, (2) investigate virus dissemination in various
tissues, virus titers in cloacal swabs, and antibody response, and (3) examine the
dynamics of interferon (IFN)-γ, interleukin (IL)-10, IL-18, and IL-8 gene expression
following FAdV-4 infection.
2. Material and methods 2.1. Virus
The FAdV-4 was isolated from tissues collected from chickens of a Canadian
broiler breeder flock showing no clinical signs of HHS. Virus isolation was performed by
the Animal Health Laboratory (AHL) at the University of Guelph. The virus was plaque
purified in our laboratory, and virus propagation and titration were performed in CH-
SAH cells as described by Alexander et al. (1998).
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2.2. Chickens and housing
All birds were white Leghorn SPF chickens. The eggs were obtained from the
Canadian Food Inspection Agency (CFIA), Ottawa, and they were hatched at the Arkell
Poultry Station. The birds were housed at the University Isolation Unit according to the
Animal Care Committee of the University of the Guelph in accordance with the Guide to
the Care and Use of Experimental Animals of the Canadian Council on Animal Care and
received food (21% unmedicated starter ration) and water ad libitum.
2.3. Pathogenicity assessment of FAdV-4 in SPF chicks
A chicken trial was conducted to evaluate the pathogenic potential of the FAdV-4
isolate. One hundred chicks were randomly divided into four groups: Group I (FAdV-4) -
oral, Group II (FAdV-4) -intramuscular (im), Group III (control) -oral, and Group IV
(control) -im. Groups I and II comprised 30 chickens each, and the negative control
groups (III and IV) comprised 20 chicks each. At 9 days of age, the chickens were tagged
with wing bands (Ketchum, ON) and blood samples were collected from all birds. Ten-
day-old chickens in Groups I and II were inoculated with 2x108 plaque forming units
(pfu) of virus per chick. The control chickens (mock-infected) received PBS. Groups I
and II were re-inoculated at 14 days of age with the same dose of virus. The chickens
were observed daily (3x) for the development of clinical signs. Three chicks from the
control groups and four chicks from the treatment groups were randomly selected for
necropsy by simple random sampling of their identification numbers stratified by cage,
using statistical software (MINITAB 14) at 0, 3, 5, 7, 14, 21, and 28 days post-infection
(d.p.i.). The chickens were euthanized according to the University of Guelph Animal
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Care Committee regulations (Isolation Unit, University of Guelph, Ontario, Canada),
necropsied, and examined for the presence of gross lesions, and tissues from thymus,
lung, heart, kidney, liver, bursa of Fabricius, and cecal tonsils were collected. Each organ
was sectioned into three portions: one was placed into 10% formalin for histology, the
second was stored at -80˚C for quantitative polymerase chain reaction (qPCR), and the
third was stored at -80˚C for virology. Cloacal swabs were collected at 0, 3, 5, 10, 14, 21,
28 d.p.i. from all chicks and their virus titers were determined in CH-SAH cells. Blood
samples were collected weekly, at 0, 7, 14, 21, and 28 d.p.i. and tested for FAdV-specific
antibodies (Abs) by enzyme-linked immunosorbent assay (ELISA) as described (Ojkic
and Nagy, 2003). The results were expressed by calculating the sample-to-positive (S/P)
ratio for each sample from mean optical density (OD405) readings.
2.4. Experimental design for determination of cytokine mRNA expression
Eighty ten-day-old chicks were randomly divided into two groups. Group I
contained 50 birds and Group II (negative control) contained 30 birds. Each bird in
Group I received 2x108 pfu/ml of the virus im. The negative control chicks received PBS
by im administration. Five chicks from the infected group and three chicks from the
uninfected group were selected randomly for necropsy at 0, 1, 2, 3, 4, 5, 6, 7, 10, and 14
d.p.i. The rationale for selecting these time points was guided by the assumption that
rarely is one immune measure at one time point sufficient for any conclusion. Therefore,
we selected several time points in order to cover the turning point of the infection. The
chickens were euthanized and the spleen, cecal tonsil, and liver were collected and
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immediately submerged in RNA stabilization reagent (RNAlater, Qiagen, Mississauga,
ON, Canada) and stored at -80°C until RNA extraction.
2.5. Quantification of viral DNA in tissues
In order to determine the viral load in the tissues, real-time PCR was performed using
SYBR Green as an intercalating dye. The FAdV-4 ORF-14 gene was used as an indicator
for the presence of viral DNA. The forward primer was 5’-
AGTGTGTATGTGCGTTGGGTAG-3’ and reverse primer was 5’-CATTGTCATAAC
GATGGTGTAG-3’. The 20 l qPCR reaction mix, containing 1 l (10 pmol) of each
primer, 10 l of QuantiTec SYBR Green I PCR Master Mix (Qiagen), 2 l DNA, and 6
l nuclease-free water, was prepared using computerized automated liquid handling
robotics CAS-1200 (Corbett Research). The qPCR program was run as a first step of 15
min at 95C to activate Taq polymerase, followed by 40 cycles of 95C for 20 sec, 55C
for 15 sec, and 72C for 20 sec, with an acquiring step at 76C for 15 sec, using the
Rotor-Gene 6000 System (Corbett Research). Quantitative measurement of the PCR
product was carried out by incorporation of the SYBR Green I fluorescent dye into
double-stranded DNA. To establish standard curves, 10-fold serial dilutions (10-1 to 10-7)
of FAdV-4 genomic DNA starting at 12.2 ng/l (2.4 x 108 copies/l) were made and used
as qPCR templates. Melting curve analysis was performed after each run to control the
specificity of the PCR products. Negative control DNA extracted from uninfected
chickens and no template control were routinely included in each experiment.
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2.6. RNA extraction and real-time PCR for determination of cytokine gene expression
Total RNA was extracted from the spleen, cecal tonsil, and liver samples by
Trizol reagent (Invitrogen, Canada Inc., Burlington, ON, Canada) as described by the
manufacturer. The tissues were homogenized in 1 ml Trizol followed by chloroform
extraction, isopropanol precipitation, and a 75% ethanol wash. The RNA pellet was
dissolved in 20 µl of RNAse-free water. The concentration of the RNA was measured by
NanoVue spectrophotometer. In order to remove possible contaminating DNA, a DNase
inactivation reagent (Invitrogen) was applied. For cDNA synthesis from total RNA,
SuperScriptTM II Reverse Trancriptase (Invitrogen) was used according to the
manufacturer’s instructions and the random primers were from Invitrogen. The cDNAs
were stored at -80°C until used for real-time PCR.
A real-time PCR assay was employed to quantify the expression of β-actin and
cytokine genes in cDNA samples. The PCR reaction was prepared in a total volume of 20
µl of Qiagen Quantitect SYBR Green PCR kit (Qiagen) containing hot start Taq
polymerase and cDNA. Each assay was performed without a template control and with
three dilution series from the standard. The sequences and other features of specific
primers for chicken IFN-γ, IL-18, IL-10, IL-8, and β-actin have been described (Abdul-
Careem et al., 2006; 2007). The primers were synthesized by the Laboratory Services,
University of Guelph. The PCR conditions for different segments of each cycle were
optimized for all target genes.
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Previous research indicated that after FAdV infection the most variability in
cytokine gene expression has been in the liver (Grgic et al., 2012). Therefore, in the liver,
mRNA expression was determined for IFN-γ, IL-18, IL-10, and IL-8, while for cecal
tonsil and spleen, mRNA expression was determined only for IFN-γ and IL-18.
2.7. Statistical analysis
In order to evaluate whether viral copy number differed among tissues, a Kruskal-
Wallis test was performed. When statistically significant P-values were identified, pair-
wise comparisons were performed between different tissues. P-values of <0.05 were
considered to be statistically significant.
Real-time PCR efficiency was calculated by 10(-1/slope of the standard curve) (Rasmussen,
2001). The mRNA expression was calculated relative to β-actin gene expression using
the equation (Etarget)ΔCPtarget(control –sample)/(Eref)
ΔCPref(control –sample) (Pfaffl, 2001). In this
equation, CP (crossing point) is the point at which the fluorescence rises significantly
above the background. The difference in cytokine expression between groups was
assessed by a Mann-Whitney test and comparisons were considered significant at P<0.05.
3. Results 3.1. Pathogenicity
Clinical signs characterized by depression, chickens huddling in corners with
ruffled feathers and exhibiting characteristic posture, with the chest and beak resting on
the ground was not seen in any groups of chicks. Distinctive gross lesions,
hydropericardium, pale, swollen and friable liver were absent. The characteristic lesions
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of HHS on histological examination described as intranuclear inclusion bodies in
hepatocytes were not detected in any of the infected chickens.
3.2. Viral genome copy number in tissues
Viral genome copy numbers in liver, bursa of Fabricius, and cecal tonsil were
determined by quantitative real-time PCR and the results over the entire study are
summarized in Table 4.1. No viral DNA was detected in any tissues from any chickens
before inoculation or in the mock-infected chickens. Each virus and each inoculation
group (oral and im) were subjected separately to the Kruskal-Wallis test to evaluate the
differences in number of viral copies among tissues. The cecal tonsil was the organ with
the highest number of viral copies, followed by liver and then bursa, irrespective of
inoculation route. At group level for FAdV-4 orally inoculated birds, there were
significant differences (P<0.001) in number of viral copies, whereas for the im group, the
differences tended to be significant (P=0.06). For the orally inoculated birds, viral copy
number was higher in cecal tonsil than in liver (P=0.0207), and bursa (P=0.0001),
whereas viral copy numbers in liver ranked only marginally higher than viral copy
numbers in bursa (P=0.0759). For the im inoculated birds, viral copy number was higher
in cecal tonsil than in the bursa (P=0.0154), whereas differences among other organs
were not significant (P>0.25).
3.3. Virus titers in feces
No virus was detected in the cloacal swabs of any chickens before inoculation nor
in the mock-infected groups at any time. The virus titers are given in Table 4.2. The
difference in titers between the groups of orally and im inoculated chickens was
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statistically significant (P<0.001) when it was tested using a 2-sample Wilcoxon test. The
oral group had higher ranks. The highest titers were found in the orally inoculated group
at days 3 p.i. and 5 p.i. Chickens shed the virus through the entire study period in both the
oral and im groups.
3.4. Antibody response
Ab against fowl adenovirus was not detected in any birds before inoculation (0
d.p.i.) nor in the mock-infected birds at any time. An Ab response against FAdV-4
appeared at 7 d.p.i. and the difference between inoculated groups and negative controls
was statistically significant (P<0.001). The inoculated chickens had higher antibody
levels than the mock-infected birds (Fig. 4.1A). Chickens inoculated im had higher
values than birds inoculated orally (P<0.001). When the same sera were tested against
FAdV-9, a heterologous virus, the results were similar, but the levels were slightly lower
(Fig. 1B).
3.5. Expression of cytokine genes
Expression of selected cytokine genes after FAdV-4 inoculation was measured in
cecal tonsil, liver and spleen.
In the cecal tonsils there were no significant differences (P>0.05) in the
expression of IFN-γ and IL-18 mRNA when compared with the values for the appropriate
uninfected controls (Fig. 4. 2). Up-regulation of IFN-γ and IL-18 mRNA was evident in
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infected birds at 1, 3 and 7 d.p.i., although the results were not statistically significant.
The mRNA expression changed after 7 d.p.i., showing a downward trend at 10 d.p.i.
The expression of IFN-γ, IL-18, IL-10, and IL-8 mRNA in the liver of FAdV-4
infected and uninfected control chickens is illustrated in Fig. 4.3. Expression of all
selected cytokines appeared to differ between infected and uninfected chickens. In the
liver, an upward trend for all the studied cytokines was recorded for the infected chickens
throughout the study period up to 10 d.p.i. There was a statistically significant increase
(P<0.05) in IFN-γ gene expression of infected chickens at 3 d.p.i. when compared to
chickens in the uninfected group. The expression pattern of IL-10 was similar to the
expression of IFN-γ, being significantly higher at 3 d.p.i. in the infected group of
chickens when compared to the uninfected group (P<0.05). The expression of IL-8 was
not significantly different (P>0.05) between infected and uninfected groups, although an
upward trend in the infected group was observed throughout the study period.
The expression of IFN-γ and IL-18 mRNA in the spleen of FAdV-4 infected and
uninfected control chickens is shown in Fig. 4. 4. Both IFN-γ and IL-18 were detected at
all time points and were down-regulated, in contrast to expression levels in cecal tonsil
and liver. When gene expression of both IFN-γ and IL-18 in the spleen was compared
between infected groups and uninfected controls, statistically significant (P<0.05) down-
regulation was observed in the infected group at 10 d.p.i.
4. Discussion
Fowl adenoviruses are associated with numerous clinical conditions, but their
primary role in disease is still not understood. Most strains of FAdVs follow the same
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pattern of infection. Following initial multiplication, viraemia occurs and results in virus
spread to virtually all organs. The main sites of virus replication are the respiratory and
alimentary systems (Saiffudin and Wilks, 1991).
In this study, despite the lack of clinical signs and gross and histological changes,
virus was found in selected tissues and cloacal swabs of all infected birds. In both the
orally and im inoculated groups, the highest numbers of viral genome copies were found
in cecal tonsil. This finding is in agreement with previous work on FAdV-8 (Grgic et al.,
2011). Ojkic and Nagy (2003) reported that replication and distribution of another FAdV
serotype in chicken tissues can be distinctly influenced by the route of virus inoculation.
Since FAdV-4 is transmitted horizontally among birds by the oral-fecal route (Cowen,
1992), the im route and the more natural oral route of infection were compared. The
results showed that the orally inoculated group had a higher number of viral copies in
general. This finding is in contrast to the previously published work related to FAdV-8
(Grgic et al., 2011), where im-inoculated chicks had a higher number of viral copies. The
reason for this difference might be explained by the fact that different serotypes were
applied in those two studies. Moreover, in the present study, virus was isolated from
cloacal swabs through the entire study period (28 d.p.i.) in the im-inoculated group, while
for the orally inoculated chickens, shedding was recorded up to 21 d.p.i. Orally
inoculated chickens exhibited a significantly higher virus titer in feces than the im-
inoculated group throughout the entire study period. This result is in agreement with the
FAdV-8 pathogenicity study, where shedding was also reported up to 28 d.p.i.
Ab response was significantly higher in im-inoculated chicks than in orally inoculated
chicks. In agreement with this finding, FAdV-8 also induced higher antibodies when the
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birds were infected via the im route (Grgic et al., 2012). Additionally, all sera were tested
with heterologous FAdV-9 virus, and while results were similar, S/P ratios were lower,
supporting the hypothesis that heterologous virus can be used to detect adenovirus
antibodies against all 12 serotypes. More importantly, we determined that FAdV-4 is
immunogenic, i.e, can induce an adequate antibody response. Currently available
vaccines against HHS are composed of infected liver homogenates or virus propagated in
chicken liver cells and inactivated with 0.5% formalin or 0.1% β-propiolactone (Anjum,
1990). However, the inactivated vaccines currently available are based on virulent
viruses, with the potential risk of a vaccine break if inactivation of the virus is
incomplete.
Furthermore, cytokine responses in liver, spleen, and cecal tonsils were
investigated subsequent to infection with an FAdV-4 isolate, showing that IFN-γ, IL-18,
IL-10 and IL-8 are activated at early times after infection. The cytokines studied here are
associated with T helper (Th)1, pro-inflammatory, and regulatory activities. In this study,
the expression of these cytokines was altered during the course of FAdV-4 infection. The
expression of cytokines has been demonstrated in a chicken hepatoma cell line (CH-
SAH) and in chicken liver, spleen, and cecal tonsil following FAdV-8 infection (Grgic et
al., 2012). However, this is the first report on the cytokine repertoire associated
specifically with FAdV-4 infection. Enhanced expression of IFN-γ in liver and cecal
tonsil was noted, with a significant increase in expression at 3 d.p.i. in the liver. IFN-γ is
considered an essential cytokine in the host defense against a variety of pathogens. It has
been shown that IFN-γ inhibits Marek’s disease virus (MDV) replication by inducing
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nitric oxide synthesis (Xing and Schat, 2000), reduces replication of coccidian parasites
(Lillehoj and Choi, 1998), and inhibits src oncogene-induced tumor growth (Plachy et al.,
1999). IFN-γ is also a key factor in inhibiting reactivation of chronic infection with
murine gammaherpesvirus (Steed et al., 2007).
IL-18 was originally identified as a factor promoting IFN-γ production by T and
NK cells when costimulated by IL-12 (Okamura et al., 1995). IL-18 is a pro-
inflammatory cytokine (Göbel et al., 2003) involved in activation of neutrophils and
natural killer (NK) cells in mammals (Okamura et al., 1995). Differential expression of
IL-18 in chickens either genetically susceptible or resistant to Marek’s disease revealed
that susceptible birds have a higher expression of IL-18 in the spleen than resistant birds
(Kaiser et al., 2003). Mice lacking the IL-18 gene demonstrate impaired clearance of
neurovirulent influenza A virus-infected neurons from the brain (Mori et al., 2001).
Efficacy of feline leukemia virus (FLV) DNA vaccine is improved by coadministration
with IL-18 expression vectors (Hanlon et al., 2001). In the present study, enhanced
expression of IL-18 was noted in both liver and cecal tonsils. Enhanced expression of IL-
18 is probably an indicator of induction of inflammation and an immune response against
the virus. This finding is in agreement with a previous report where an increase in IFN-γ
and IL-18 expression was observed following FAdV-8 infection (Grgic et al., 2012).
Enhanced expression of IL-10 in the liver was recorded throughout the entire
study period, with significant up-regulation at 3 d.p.i. in the infected group. Our finding
that IL-10 was up-regulated following FAdV-4 infection is in agreement with a previous
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report (Grgic et al., 2012), and may indicate an immune evasive mechanism used by the
virus to skew the immune response, supporting persistence of the virus (Grgic et al.,
2006). IL-10 has both immunosuppressive and immunostimulating effects on cells of the
adaptive immune system (O’Garra and Vieira, 2007). Simultaneous expression of IFN-γ
and IL-10 also has been reported after infection with porcine reproductive and respiratory
syndrome virus (Johensen et al., 2002). More recently Parvizi et al. (2009) reported
persistent up-regulation of IFN-γ and IL-10 in a MDV-susceptible line of chickens. In
humans, up-regulation of both IFN-γ and IL-10 is directly related to IL-12 stimulation of
CD4+ and CD8+ T cells (Gerosa et al., 1996). IL-10 is a potent stimulator of NK cells, a
function that probably helps clear pathogens. Enhanced expression of IL-10 may be
induced by the chicken immune system in order to regulate the immune response and
inflammation caused by the virus.
Cytokines are considered important mediators of inflammation and regulators of
the immune response. The first defense against viral infection is an inflammatory
response, including release of inflammatory cytokines. IL-8 is a potent neutrophil and T
lymphocyte chemotactic factor released by a variety of mammalian cells (Oppenheim et
al., 1991), and according to Bruder and Kovesdi (1997) adenovirus infection triggers
Raf/mitogen-activated protein kinase pathways that induce synthesis of IL-8 and thus
contribute to the early inflammatory response. Increased expression of IL-8 in the present
study may be a reactive response of the host to reduce the viral load.
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In summary, in this study, the FAdV-4 virus was found in tissues and cloacal swabs from
the inoculated chickens and a strong antibody response was induced. However, no
clinical signs or gross or histological changes were detected. FAdV-4 infection was
associated with increased expression of IL-18, IFN-γ, IL-10 and IL-8 in liver and
increased expression of IL-18 and IFN-γ in cecal tonsil, providing useful information
about important mediators involved in immunity against HHS virus infection. Although
we have gained further insight into FAdV-associated cytokine gene expression, the
mechanisms and molecular events governing the production of cytokines during infection
still remain to be described. Finally, this study demonstrated that this FAdV-4 isolate
under experimental conditions was non-pathogenic and could be used as live vaccine
against HHS.
Acknowledgements
This work was supported by the Natural Sciences and Engineering Research
Council of Canada (NSERC), the Poultry Industry Council (PIC) of Ontario and the
Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA). The authors thank
the Isolation Unit personnel for their professional animal care and assistance.
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Table 4.1. Numbers of positive tissues and viral genome copy numbers in tissues of chickens inoculated with FAdV-4.
* = viral genome copy number/10 µg of total tissue DNA was determined by qPCR § = number of positive tissues/number analyzed tissues
days p.i.
Inoculation route
tissue oral im
Liver 3.4x100* 3/4§ 7.5x100 2/4
3 Bursa 5.6x100 2/4 5x100 1/4
Cecal tonsil 2.6x101 4/4 5.3x102 4/4
Liver 1.4x102 3/4 8.3x100 4/4
5 Bursa 6.1x100 4/4 1.2x101 2/4
Cecal tonsil 5.7x102 4/4 1.3x102 4/4
Liver 2.2x100 2/4 2.3x100 2/4
7 Bursa 0x100 0/4 6x100 2/4
Cecal tonsil 2.4x101 1/4 3.5x100 3/4
Liver 2.8x100 4/4 5.9x100 2/4 14 Bursa 4.9x100 3/4 4.6x100 2/4
Cecal tonsil 1.2x101 4/4 8.6x100 4/4
Liver 1.1x101 4/4 2.4x101 4/4
21 Bursa 2.3x101 3/4 3.1x102 4/4
Cecal tonsil 6.4x103 4/4 3.6x102 3/4
Liver 6.5x102 4/4 3.5x102 4/4 28 Bursa 1.4x100 2/4 7.4x101 1/4
Cecal tonsil 1.7x102 4/4 6.5x102 4/4
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Table 4.2. Virus titers in the feces of chickens inoculated with FAdV-4.
Days Inoculation route post-infection Mean titers (pfu/ml) in swabs (log10 mean ± log10SEmean)
Chickens shedding the virus (%)†
Oral Im 9.9x103 (3.30±0.22) 1.4x103 (0.69±0.24)
3 P≤0.001 (93) (25) 1.2x104 (3.15±0.32) 1.2x103 (1.01±0.33)
5 P≤0.001 (83) (32)
1.3x102 (0.24±0.17) 6.8x102 (0.54±0.31)
10 P=0.49 (13) (20)
3.9x101 (0.49±0.23) 2.5x101 (0.15±0.10)
14 P=0.26 (27) (13)
3.3x101 (0.20±0.14) 2.5x101 (0.34±0.23)
21 P=0.25 (17) (17)
0 1.2x101 (0.12±0.12)
28 P=0.34 (0) (12)
The virus titers were determined by plaque assay.
P values < 0.05 are statistically significant. †30 chickens at day 0; 26 and 22 chickens at days 3 and 5, respectively; 18 and 14 chickens at days 10 and 14, respectively; 10 and 6 chickens at days 21 and 28, respectively.
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Figure 4.1. Antibody response to viral proteins in chickens inoculated with FAdV-4 by the oral and intramuscular routes and mock infected as measured by S/P ratios. Serum samples were collected at 7, 14, 21 and 28 days post-infection. Dark gray and white columns represent im and orally inoculated chickens, respectively. Ligth gray and black columns represent mock-infected chickens. The error bars correspond to 95% confidence intervals. Significant differences among mock infected and infected birds are indicated by asterisks (*). Panel A. Serum samples were tested against homologous virus, FAdV-4.
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Figure 4.1. Panel B. Serum samples were tested against heterologous virus, FAdV-9.
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Figure 4.2. Cytokine mRNA expression in cecal tonsil of chickens infected with FAdV-4. The groups were as follows: Uninfected (negative control) chickens, and FAdV-4 infected chickens. Target and reference gene expression in cecal tonsil was quantified by real-time RT-PCR using SYBR Green. Target gene expression is presented relative to β-actin expression and normalized to a calibrator. The results are diagrammed as whisker boxes with medians. Boxes represent interquartile ranges and whiskers indicate extreme values. The difference in cytokine expression between groups was assessed by Mann-Whitney test and comparisons were considered significant at P≤0.05 (*). ○, ● symbols represent values which were identified as outliers. Numbers 1, 3, 5, 7, and 10 represent days post infection when samples were collected.
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Figure 4.3. Cytokine mRNA expression in liver of chickens infected with FAdV-4. The groups were as follows: Uninfected (negative control) chickens, and FAdV-4 infected chickens. Target and reference gene expression in liver was quantified by real-time RT-PCR using SYBR Green. Target gene expression is presented relative to β-actin expression and normalized to a calibrator. The results are diagrammed as whisker boxes with medians. Boxes represent interquartile ranges and whiskers indicate extreme values. The difference in cytokine expression between groups was assessed by Mann-Whitney test and comparisons were considered significant at P≤0.05 (*). ○, ● symbols represent values which were identified as outliers. Numbers 1, 3, 5, 7, and 10 represent days post infection when samples were collected.
121
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Figure 4.4. Cytokine mRNA expression in spleen of chickens infected with FAdV-4. The groups were as follows: Uninfected (negative control) chickens, and FAdV-4 infected chickens. Target and reference gene expression in spleen was quantified by real-time RT-PCR using SYBR Green. Target gene expression is presented relative to β-actin expression and normalized to a calibrator. The results are diagrammed as whisker boxes with medians. Boxes represent interquartile ranges and whiskers indicate extreme values. The difference in cytokine expression between groups was assessed by Mann-Whitney test and comparisons were considered significant at P≤0.05 (*). ○, ● symbols represent values which were identified as outliers. Numbers 1, 3, 5, 7, and 10 represent days post infection when samples were collected.
*
*
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CHAPTER 5 Molecular characterization of the fiber gene of fowl adenovirus serotypes 8 and 11
Helena Grgić1, Davor Ojkić2, Éva Nagy1*
1Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, N1G 2W1, Canada. 2Animal Health Laboratory, University of Guelph, Guelph, Ontario, N1G2W1, Canada
*Corresponding author: Éva Nagy E-mail: [email protected] Tel: +1 519 824 4120 ext 54783 FAX: +1 519 824 5930
The GenBank accession numbers of the sequences reported in this paper are:
BankIt1493149 Fiber JQ034214 BankIt1493157 Fiber JQ034215 BankIt1493162 Fiber JQ034216 BankIt1493466 Fiber JQ034217 BankIt1493468 Fiber JQ034218 BankIt1493469 Fiber JQ034219 BankIt1493470 Fiber JQ034220 BankIt1494294 Fiber JQ034221 Author’s contributions
HG designed and performed experiment, carried out analysis and wrote the paper. DO
provided viruses for the research and revised manuscript. EN provided guidance during
the research and critically revised manuscript
Short communication to be submitted to Virus Genes
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Abstract
The fiber protein as one of the major constituents of the adenoviral capsid is
involved in virus entry and it has been implicated in the variation of virulence of fowl
adenoviruses (FAdVs). The most commonly isolated FAdVs in Canada belong to
serotype 8 and 11, according to current surveillance data. Therefore, four FAdV-8 and
four FAdV-11 isolates were selected to examine the possible role of the fiber gene
sequence in FAdV virulence. On the basis of the established fiber structural model, the
FAdV fiber sequence can be divided into tail, shaft and head domains comprising some
specific features. The conserved ‘RKRP’ (aa 17 to aa 20) motif fits the consensus
sequence predicted for the NLS, while the ‘VYPF’ (aa 53 to aa 56) sequence motif is
suggested to be involved in the penton base interaction. Similarly to mammalian
adenoviruses, 17 pseudorepeats have been detected in the FAdV-8 fiber shaft region, with
each repetition comprising 16 aa on average. The shaft regions of FAdV-11 fibers consist
of 20 pseudorepeats and each repetition comprising 18 aa on average. There was a 144 nt
difference between the fiber genes of the two FAdV serotypes. In the shaft region the
characteristic well-conserved TLWT motif that marks the beginning of the fiber head
domain was not evident among examined FAdVs. The FAdV-11 isolates had 99.3% aa
sequence identity and 99.1% similarity to each other and there was no conserved aa
substitution within the fibers The FAdV-8 fiber proteins showed an overall 89% aa
sequence identity and 93.4% similarity to each other and 21 nonsynonymous mutations
were detected. Virulence markers were not detected in the analyzed fiber genes of the
two FAdV serotypes.
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Keywords:
FAdV-8; FAdV-11; fiber gene sequences; conserved TLWT motif; virulence markers.
Abbreviations: FAdV-8, fowl adenovirus serotype 8; FAdV-11, fowl adenovirus
serotype 11; NLS, nuclear localization signal.
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Members of the family Adenoviridae are non-enveloped double-stranded DNA
viruses. The 12 serotypes of fowl adenoviruses (FAdV) are grouped into five species
(FAdV-A toFAdV-E) on the basis of restriction fragment length polymorphism (RFLP)
profiles and cross-neutralization (Benkő et al., 2005). Infection with several FAdV
serotypes is associated with diseases such as inclusion body hepatitis (IBH) and
infectious hydropericardium syndrome. The outcome of FAdV infection is sometimes
influenced by concurrent infection with immunosuppressive agents such as infectious
bursal disease or chicken anemia viruses (Fadly et al., 1975). However, significant
economic losses have also been encountered in the absence of co-infection with
immunosuppressive agents (Saifuddin and Wilks, 1990). The pathogenicity of FAdVs
differs even among strains of the same serotype. According to current surveillance data,
in Canada the most commonly isolated FAdVs belong to serotype 8 and 11 (Ojkic et al.,
2008a, b). It has been reported that the virulence of FAdV-8 is determined by the fiber
protein alone (Pallister et al., 1996). Variations in the fiber gene sequences have also
been implicated in the differences in tissue tropism and virulence of the canine
adenoviruses (Rasmussen et al., 1995). The objective of this study was to examine the
possible role of the fiber gene in FAdV virulence by analysis of sequences from
pathogenic and non-pathogenic FAdV-11 and FAdV-8 isolates.
Four FAdV-11 (species Fowl adenovirus D) and four FAdV-8 (species Fowl
adenovirus E) isolates were kindly provided by the Animal Health Laboratory (AHL),
University of Guelph. Two of the serotype 11 and two of the serotype 8 viruses were
isolated from flocks with clinical signs of IBH, and IBH was confirmed as the final
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diagnosis. The other four viruses were isolated from flocks with no clinical signs of IBH
(Table 5.1). The genotyping of the isolates was determined by sequence analysis of the
hexon gene as described before (Ojkic et al., 2008). The viruses were plaque purified and
propagated in chicken hepatoma (CH-SAH) cells as described (Alexander et al., 1998).
CH-SAH cells were infected at a multiplicity of infection of 0.5. Cells and supernatants
were collected when extensive cytopathic effect was observed. Virus purification and
viral DNA preparation were performed as described (Ojkic and Nagy, 2001).
FAdV-8 fiber genes were amplified by PCR with the primer set: FibFor8 (5’-CTG
GTG AAG TCC CTC TCC GC –3’) and FibRev8 (5’-GAC ATA GGG GAA CAT CAA
–3’) (Grgic et al., 2011). For FAdV-11 the primers were designed based on a comparison
of the sequences of the fiber genes of FAdV-9 and FAdV-2, since these viruses are in the
same species and they have a close genetic relationship (Corredor et al., 2008). The
following primer set was used to amplify FAdV-11 fiber genes: FibFor11 (5’-CAC TCG
CCC TAG CTT ATG-3’) and FibRev11 (5’-TGT TAA CAA AGG TCG ACT G –3’).
PCR was performed with 35 cycles of 94˚C for 30 sec, 58˚C (for FAdV-8) or 52˚C (for
FAdV-11), and 72˚C for 30 sec. The PCR products were sequenced by the Laboratory
Services, University of Guelph. Sequence assembly was performed with Vector NTI
AdvanceTM11. The coded proteins were identified using BLAST analysis with the
GenBank database and degree of identities and similarity was identified using FASTA3
(EMBL-EBI).
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The role of fiber protein in virulence of adenoviruses is not well understood,
therefore we compared the fiber sequences of serotypes 8 and 11 FAdVs isolated from
clinical submissions to the Animal Health Laboratory and originated from cases with or
without the clinical diagnosis of inclusion body hepatitis. Some FAdV serotypes have
two fiber genes, such as FAdV-1 and FAdV-4 (Chiocca et al., 1996; Griffin and Nagy,
2011), however there was only one fiber gene for all studied viruses of both serotypes.
The FAdV-8 and FAdV-11 fiber genes encoded from 524 to 525 amino acids (aa) and
572 aa, respectively.
On the basis of the established fiber structural model for mastadenoviruses (Green
et al., 1983) the FAdV-8 and FAdV-11 fiber sequences can also be divided into tail, shaft
and head domains and have some specific features.
For both serotypes the first 63 N-terminal aa with conserved clusters make up the
tail region (Fig.5.1). These conserved patterns were identical for both serotypes 8 and 11
viruses. Most adenovirus fiber sequences contain the nuclear localization signal (NLS).
Moreover, all of the known nuclear localization signals are rich in basic aa residues. A
potential NLS sequence with stretch of positively charged aa was identified within the
predicted FAdV-8 and FAdV-11 sequences. The KRAR (aa 2 to aa 5) sequence
conserved among fibers from several human adenovirus serotypes, such as HAdV-5,
HAdV-3, and HAdV-7 (Chroboczek and Jacrot, 1987; Hong et al., 1988; Signas et al.,
1985) was not detected for FAdV-8 and FAdV-11 isolates. However, a similar motif
‘RKRP’ (aa 17 to aa 20) was present in all analyzed FAdV fibers of both serotypes. The
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‘RKRP’ motif could have an NLS function that is essential for the targeting of the fiber to
the nucleus and fits the consensus sequence predicted for the NLS by PSORT II finder
for NLS. Another well conserved motif of the tail region, the ‘VYPY’ sequence found in
many mammalian adenovirus fiber proteins is most likely involved in the penton base
interaction (Caillet-Boudin, 1989; Hong and Boulanger, 1995). Equivalent sequence
motif in FAdV fiber tails of both serotypes FAdV-8 and 11 is ‘VYPF’ (aa 53 to aa 56)
(Fig.5.1). Similar motif has been reported in the fiber tail of ovine adenovirus (Vrati et
al., 1995), egg drop syndrome virus (Hess et al., 1997), and bovine adenovirus 7 (Li and
Tikoo, 2002). Interestingly, Hess et al. (1995) reported a ‘VYPY/F’ sequence in the tail
region of both FAdV-1 fibers, long and short. In Fig.5.1 we have also indicated the poly
(G) stretch found in the tail domain, though the poly (G) might be only the connection
between the tail and shaft domains without belonging to either. A poly (G) stretch has
been reported as part of the long fiber tail region of FAdV-1 (Hess et al., 1995), while the
same stretch was absent in the FAdV-10 fiber gene (Sheppard et al., 1998).
Similar to mammalian adenoviruses (Green et al., 1983; van Raaij et al., 1999),
17 repetitive elements (pseudorepeats) have been identified in the FAdV-8 fiber shaft
region, with each repetition comprising 16 aa on average (Fig. 5.2). The shaft regions of
FAdV-11 fibers consist of 20 pseudorepeats with an average of 18 aa each (Fig. 5.2). It is
important to note that in most mastadenovirus fiber shafts the last pseudorepeat before
the head domain contains a conserved amino acid sequence (KLGXGLXFD/N). This
conserved sequence was absent in both FAdV-8 and -11 fibers and has not been reported
for FAdV-1 and FAdV-10 (Hess et al., 1995; Sheppard et al., 1998).
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In human adenoviruses there is a characteristic well-conserved TLWT sequence
in the first β-strand of the head (Xia et al., 1994) that marks the beginning of the fiber
head domain. This typical knob sequence was absent in the examined FAdV-8 and
FAdV-11 sequences and was also absent in the reported FAdV-1, FAdV-4 and FAdV-10
sequences (Hess et al., 1995; Mase et al., 2010; Sheppard et al., 1998) making it
impossible to define the actual start of the head domain. The absence of TLWT motif has
also been reported for the fiber proteins of the OAV287 (Vrati et al., 1995), BAV7 (Li
and Tikoo, 2002), and EDSV (Hess et al., 1997). Further analysis of the C-terminal knob
domains has shown no homology with the mammalian fiber heads. Nevertheless, the C-
terminal 154 aa of the FAdV fiber sequences probably constitutes the head region.
The FAdV-11 isolates had 99.3% aa sequence identity and 99.1% similarity to
each other. There was no conserved amino acid substitution within the fibers of the
serotype 11 viruses. The complete aa sequence alignment of the four FAdV-11 fibers and
FAdV-1 short and long fibers using FASTA3 (EMBL-EBI) method revealed 27.3%
identity and 42.9% similarity for the short fiber while for the long fiber the percent
identity and similarity was only 18.6 and 27.2%, respectively (Table 5.2). The FAdV-11
fiber protein and FAdV-9 fiber protein, which belongs to the same species group showed
an overall 84.1% aa sequence identity and 91.6% similarity. Very low 19.1% identity
and 29.1% similarity, has been observed when the fiber protein sequences of FAdV-11
isolates were compared to the FAdV-4 fiber protein that belongs to species group C
(Table5. 2). When the aa sequences of the fiber proteins of species D and E were
compared there were 47 to 50 aa differences between the fiber proteins, showing an
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overall 51.4% identity and 64.5% of similarity (Table 5.2). The fiber proteins of the
analyzed four FAdV-8 isolated showed an overall 89% aa sequence identity and 93.4%
similarity to each other. In total, 21 nonsynonymous mutations were detected within the
fiber of FAdV-8 isolates from birds with signs of IBH compared to the isolates from non-
IBH cases (Table 5.3). Nonsynonymous mutations are highlighted in Table 5.3. Because
of such a large number of nonsynonymous mutations it is difficult to speculate about the
effect of these mutations on the pathogenicity of these isolates. Further, comparison of
the FAdV-8 fibers and the FAdV-1 short and long fibers revealed 28.2% identity and
41.8% similarity for the short, while for the long fiber the percent identity and similarity
was only 19.2% and 27.4%, respectively (Table 5. 2). When the Ontario FAdV-8 fiber
proteins were compared to the mildly virulent CFA3 and hypervirulent CFA40 (Pallister
et al., 1996) isolates, the percent identity and similarity on average was 70.8/74.2 and
65.8/69.9, respectively (Table 5.2). The significantly decreased identity and similarity
was due to difference in size, the Ontario isolates were on average 96 aa longer.
Moreover, the last 65 aa representing 15% of the fiber protein of CFA40 and CFA3
showed the highest divergence compared to the Ontario fiber proteins. Interestingly, the
highest divergence was in the knob region. These results are different from the data
published by Mase et al. (2010) who showed that nt sequences of Japanese FAdV-4
isolates were identical to each other and had 98.5% homology with isolates from India
and Pakistan. Similarly, FAdV-1 isolates from 4 outbreaks of gizzard erosions in broilers
shared 100% identity at the protein level. Moreover, the short fiber of the pathogenic
isolates was 100% identical to the apathogenic Ote strain (Marek et al., 2010).
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We have shown that FAdV-8 and FAdV-11, representing species E and D have
only one fiber gene. There was a 144 nt difference between the fiber genes of the two
FAdV serotypes, mapped to the shaft region. According to our data virulence might not
be associated only with sequences of the fiber gene. In order to identify the critical sites
responsible for the pathogenicity of FAdVs further molecular analyses would be required.
Moreover, to estimate the influence of adenoviral fiber genetics on pathogenesis,
experimental infections would be necessary.
Acknowledgements
This work was supported by the Natural Sciences and Engineering Research
Council of Canada (NSERC), the Canadian Poultry Research Council (CPRC), and the
Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA).
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Table 5.1. Description of the isolates.
* = FAdV-8 isolate from the Chapter 2
Designation AHL code Pathogenic/Non-pathogenic GenBank #
FAdV-8-ON-P1 04-53357-125 IBH case BankIt1493157 Fiber JQ034215
FAdV-8-ON-P2 06-41265-07 IBH case BankIt1493162 Fiber JQ034216
FAdV-8-ON-NP1 05-50052-3180 Non-IBH case BankIt1494294 Fiber JQ034221
FAdV-8-ON-NP2 06-16340-336-OH Non-IBH case BankIt1493149 Fiber JQ034214
FAdV-8* N/A IBH case - non-pathogenic HG-GU734104
FAdV-11-ON-P1 06-25854-1 IBH case BankIt1493469 Fiber JQ034219
FAdV-11-ON-P2 05-17766 IBH case BankIt1493470 Fiber JQ034220
FAdV-11-ON-NP1 05-50052-2924-1 Non-IBH case BankIt1493466 Fiber JQ034217
FAdV-11-ON-NP2 05-50052-3181 Non-IBH case BankIt1493468 Fiber JQ034218
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Table 5.2. Percent identities/similarities between FAdV-8 and FAdV-11 fiber proteins and their homologues.
S= short fiber, L= long fiber
FAdV-8/E (U40587) = CFA 40 hypervirulent, FAdV-8/E (U40588) = CFA 3 mildly virulent
Percent identities and similarities were calculated using FASTA3 (EMBL-EBI).
IsolatesType/Accession FAdV-8-ON-
P1 FAdV-8-ON-
P2 FAdV-8-ON-
NP1 FAdV-8-ON-
NP2 FAdV-11-
ON-P1 FAdV-11-
ON-P2 FAdV-11-ON-NP1
FAdV-11-ON-NP2
FAdV-8-ON-P1 - 86.3/92 94.3/96.8 92.2/94.9 - - - -
FAdV-8-ON-P2 - - 82.3/90.1 81.4/88.8 - - - -
FAdV-8-ON-NP1 - - - 97.7/97.7 - - - -
FAdV-8-ON-NP2 - - - - - - - -
FAdV-8 (U40587) 67/71.9 66.5/69.2 66.5/71.5 64/68.5 38.6/49.6 40/50.8 40/50.8 40/50.8
FAdV-8 (U40588) 75.3/77.2 67.4/72.3 67.4/72.3 75.3/76.5 42.2/52.6 43.6/53.8 43.6/53.8 43.6/53.8
FAdV-8 (GU734104) 86.5/92.2 99.8/99.6 83.1/90.3 81.6/89 49.2/63.7 50.6/64.9 50.6/64.9 50.6/64.9
FAdV-11-ON-P1 51.6/64.2 52.9/65.4 52.9/65.4 52.9/65.4 - 97.2/97 97.2/97 100/99.8
FAdV-11-ON-P2 50.9/63.3 52.2/64.5 52.2/64.5 52.2/64.5 - - 100/99.8 100/99.8
FAdV-11-ON-NP1 51.3/65.1 52.7/66.3 52.7/66.3 52.7/66.3 - - - 100/99.8
FAdV-11-ON-NP2 49/63.6 50.3/64.8 50.3/64.8 50.3/64.8 - - - -
FAdV-1/S (AA46933) 29.2/41.9 29/41.4 28.7/42.1 28.3/41.8 27/42.6 27.4/43 27.4/43 27.4/43
FAdV-1/L (AA46933) 19.5/27.9 17.9/25 19.7/28.5 19.8/28.3 18.6/27.2 18.6/27.2 18.6/27.2 18.6/27.2
FAdV-4/1 (GU188428) 24.3/36.2 24.7/34.8 23.7/34.9 23.4/34.5 18.8/28.9 19.2/29.2 19.2/29.2 19.2/29.2
FAdV-4/2 (GU188428) 39.3/54.1 38/54.1 39.4/53.4 38.8/52.8 35.9/51.6 35.4/49.3 35.4/49.3 35.4/49.3
FAdV-9 (AF083975) 52.4/67.3 52.5/66.5 52.8/67 52.8/66.1 82/89.7 84.8/92.3 84.8/92.3 84.8/92.3
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Table 5.3. Differences in amino acids between FAdV-8 isolates derived from flocks with or without the clinical signs or IBH.
Amino acids at position
Origin of viruses: 374 375 390 408 443 444 452 458 468 471 472 473 474 477 484 494 503 506 513 522 523
No signs of IBH P S A E V D G S V N A T I Q S S S L Y D V
Signs of IBH A A/N E/T Q L E D - C T V R V R L A/T A M F N A
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FAdV-8 FAdV-8-ON-NP1 MATSTPHAFSFGQIGSRKRPAGGDGERDASKVPKMQTPAPSATANGNDELDLVYPFWLQN 60 FAdV-8-ON-NP2 MATSTPHAFSFGQIGSRKRPAGGDGERDASKVPKMQTPAPSATANGNDELDLVYPFWLQN 60 FAdV-8-ON-P2 MATSTPHAFSFGQIGSRKRPAGGDGERDASKVPKMQTPAPSATANGNDELDLVYPFWLQN 60 FAdV-8-ON-P1 MATSTPHAFSFGQIGSRKRPAGGDGERDASKVPKMQTPAPSATANGNDELDLVYPFWLQN 60 ************************************************************ FAdV-8-ON-NP1 GSTGGGGGGG 70 FAdV-8-ON-NP2 GSTGGGGGGG 70 FAdV-8-ON-P2 GSTGGGGGGG 70 FAdV-8-ON-P1 GSTGGGGGGG 70 ********** FAdV-11 FAdV-11-ON-NP1 MAKSTPFAFSMGQHSSRKRPADSENTQNASKVAKTQTSATRAGVDGNDDLNLVYPFWLQN 60 FAdV-11-ON-NP2 MAKSTPFAFSMGQHSSRKRPADSENTQNASKVAKTQTSATRAGVDGNDDLNLVYPFWLQN 60 FAdV-11-ON-P2 MAKSTPFAFSMGQHSSRKRPADSENTQNASKVAKTQTSATRAGVDGNDDLNLVYPFWLQN 60 FAdV-11-ON-P1 MAKSTPFAFSMGQHSSRKRPADSENTQNASKVRQNADFCHARRCRRKYDLNLVYPFWLQN 60 ******************************** :. . : ************ FAdV-11-ON-NP1 STSGGGGGG 69 FAdV-11-ON-NP2 STSGGGGGG 69 FAdV-11-ON-P2 STSGGGGGG 69 FAdV-11-ON-P1 STSGGGGGG 69 ********* Figure 5.1. Amino acid sequence alignment of the tail sequence of FAdV-8 and FAdV-11 isolates made by CLUSTALW. The yellow arrow indicates motif which has NLS function. The blue arrow shows conserved sequence motif found in mammalian adenovirus tail region involved in penton base interaction. The green arrow shows poly (G) stretch which is connection between tail and shaft region.
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MGTSTPHAFSFGQIGSRKRPAGGDGERDASEVPKMETPAPSAT ANGNDELDLVYPFWLQNGSTGGGGGGGSGGN Tail
β βββ βββ βββ β 1 PS LNP PFL DP NGP LTVQ 2 NN LLK VNT TA PIA VTN 3 KA LTL SYD PE SLE LTNQ 4 QQ LAV KID PE GPL KATT 5 EG IQL SVD PT TLE V 6 DD VDW ELT VK LDP NGPLDSSA 7 TG ITV RVD DT LLV EDDGSGQG 8 KE LGV HLN PD GPI TADQ Shaft 9 NG LDL EID NQ TLK ITPGSA
10 GG VLS VQL KP QGG LNSQS 11 DG IQV VTQ NS IEV DN 12 GA LDV KVV AN GPL STTP 13 NG LTL NYD TG DFT VNA 14 GT LSI LRN PS LVA NAYLTSGA 15 RT LQQ FTA KG ENS SQFSFP 16 CA YYL QQW LS DGL IF17 SS LYL KLD RT RFT
GMSSDPSYQNARYFTFWVGGGAAMNLSQLSTP TITPSTTEWTAFAPALNYSGAPAFVYDANPVF TIYFQPNTGRVDTYLPVLTGDWKTSSTYNPGT Head ITLVVRNATIQLQSQSTFTTSVCYNFRCQNSGIF NNNATSGTLTLGPIFYSCPALSTADVS
Figure 5. 2. Regions of FAdV-8 and FAdV-11 fibers. Bold indicates hydrophobic amino acids. Residues forming β strands are indicated above pseudorepeats. Shaft region pseudorepeats are numbered on the left side.
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MAKSTPFAFSMGQHSSRKRPADSENTQNASKVAKTQTSATR AGVDGNDDLNLVYPFWLQNSTSGGGGGGSGGN Tail
β βββ βββ βββ β 1 PS LNP PFI DP NGP LYVQ 2 NS LLY VKT TA PIE VEN 3 KS LAL AYD SS LDV DAQ 4 NQ LQV KVD AE GPI RISP 5 DG LDI AVD PS TLE VD 6 DE WEL TVK LD PAG PISSSS 7 AG INI RVD DT LLI EDDDT 8 AQ VKE LGV HL NPN GPITADQ 9 DG LDL EVD PQ TLT VTTSGAT 10 GG VLG VLL KP SGG LQTSI Shaft 11 QG IGV AVA DT LTI SSNT 12 GT VEV KTD PN GSI GSSS 13 NG IAV VTD PA GPL TTSS 14 NG LSL KLT PN GSI QSSS 15 TG LSV QTD PA GPI TSGA 16 NG LSL SYD TS DFT VSQ 17 GM LSI IRN PS AYP DAYLESG 18 TN LLN NYT AY AEN S 19 SN YKF NCA YF LQS WYS 20 NG LVT SSL YL KIN RDN
LTSLPSGQLSENAKYFTFWVPTYESMNLSNVAT PTITPSSVPWGAFLPAQNCTSNPAFKYYLTQP PSIYFEPESGSVQTFQPVLTGDWDTNTYNPGTV Head QVCILPQTVVGGQSTFVNMTCYNFRCQNPGIF KVAASSGTFTIGPIFYSCPTNKLTQP
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Figure 5.3. Supplementary data. Amino acid alignment of FAdV-8 fibers derived from flocks with and without signs of IBH. Highlighted amino acids represent conserved substitutions. Symbol “:” means that conserved substitutions have been observed while, “.” means that semi-conserved substitutions are observed. FAdV-8-ON-NP1 MATSTPHAFSFGQIGSRKRPAGGDGERDASKVPKMQTPAPSATANGNDELDLVYPFWLQN 60 FAdV-8-ON-NP2 MATSTPHAFSFGQIGSRKRPAGGDGERDASKVPKMQTPAPSATANGNDELDLVYPFWLQN 60 FAdV-8-ON-P1 MATSTPHAFSFGQIGSRKRPAGGDGERDASKVPKMQTPAPSATANGNDELDLVYPFWLQN 60 FAdV-8-ON-P2 MATSTPHAFSFGQIGSRKRPAGGDGERDASKVPKMQTPAPSATANGNDELDLVYPFWLQN 60 ************************************************************ FAdV-8-ON-NP1 GSTGGGGGGG-SGGNPSLNPPFLDPNGPLAVQNNLLKVNTAAPITVTNKALTLAYEPDSL 119 FAdV-8-ON-NP2 GSTGGGGGGG-SCGNPSLIPPFLDPNGPLTVKNNLLKVNTAAPITVTNKALTLAYEPDSL 119 FAdV-8-ON-P1 GSTGGGGGGG-SGGNPSLNPPFLDPNGPLAVQNNLLKVNTAAPITVTNKALTLAYEPDSL 119 FAdV-8-ON-P2 GSTGGGGGGGGSGGNPSLNPPFLDPNGPLAVQNNLLKVNTAAPITVANKALTLAYEPDSL 120 ********** * ***** **********:*:**************:************* FAdV-8-ON-NP1 ELTNQQQLAVKIDPEGPLKATTEGIQLSVDPTTLEVDDVDWELTVKLDPDGPLDSSATGI 179 FAdV-8-ON-NP2 ELTNQQQLAVKIDPEGPLKATTEGIQLSVDPTTLEVDDVDWELTVKLDPDGPLDSSATGI 179 FAdV-8-ON-P1 ELTNQQQLAVKIDPEGPLKATTEGIQLSVDPTTLEVDDVDWELTVKLDPDGPLDSSATGI 179 FAdV-8-ON-P2 ELTNQQQLAVKIDPEGPLKATTEGIQLSVDPTTLEVDDVDWELTVKLDPDGPLDSSATGI 180 ************************************************************ FAdV-8-ON-NP1 TVRVDETLLVEDDGSGQGKELGVHLNPDGPITADQNGLDLEIDNQTLKITPGSAGGVLSV 239 FAdV-8-ON-NP2 TVRVDETLLVEDDGSGQGKELGVHLNPDGPITADQNGLDLEIDNQTLKITPGSAGGVLSV 239 FAdV-8-ON-P1 TVRVDETLLVEDDGSGQGKELGVHLNPDGPITADQNGLDLEIDNQTLKITPGSAGGVLSV 239 FAdV-8-ON-P2 TVRVDETLLIEDVGSGQGKELGVNLNPTGPITADDQGLDLEIDNQTLKVNSVTGGGVLAV 240 *********:** **********:*** ******::************:.. :.****:* FAdV-8-ON-NP1 QLKPQGGLNSQSDGIQVVTQNSIEVDNGALDVKVVANGPLSTTPNGLTLNYDTGDFTVNA 299 FAdV-8-ON-NP2 QLKPQGGLNSQSDGIQVVTQNSIEVDNGALDVKVVANGPLSTTPNGLTLNYDTGDFTVNA 299 FAdV-8-ON-P1 QLKPQGGLNSQSDGIQVVTQNSIEVDNGALDVKVVANGPLSTTPDGLTLNYDTGDFTVNA 299 FAdV-8-ON-P2 QLKSQGGLTAQTDGIQVNTQNSITVTNGALDVKVAANGPLESTDTGLTLNYDPGDFTVNA 300 ***.****.:*:***** ***** * ********.*****.:* *******.******* FAdV-8-ON-NP1 GTLSILRNPSLVANAYLTSGARTLQQFTAKGENSSQFSFPCAYYLQQWLSDGLIFSSLYL 359 FAdV-8-ON-NP2 GTLSILRNPSLVANAYLTSGARTLQQFTAKGENSSQFSFPCAYYLQQWLSDGLIFSSLYL 359 FAdV-8-ON-P1 GTLSILRNPSLVANAYLTSGASTLQQFTAKGENSSQFSFPCAYYLQQWLSDGLIFSSLYL 359 FAdV-8-ON-P2 GTLSIIRDPALVANAYLTSGASTLQQFTAKSENSSQFSFPCAYYLQQWLSDGLVLSSLYL 360 *****:*:*:*********** ********.**********************::***** FAdV-8-ON-NP1 KLDRTRFTGMSSDPSYQNARYFTFWVGGGAAMNLSQLSTPTITPSTTEWTAFAPALNYSG 419 FAdV-8-ON-NP2 KLDRTRFTGMSSDPSYQNARYFTFWVGGGAAMNLSQLSTRTITPSPPEWMAFAPALNYSG 419 FAdV-8-ON-P1 KLDRTRFTGMSSDAAYQNAKYFTFWVGGGEAMNLSQISTPTITPSTTQWNAFAPALNYSG 419 FAdV-8-ON-P2 KLDRAQFTNMPTGANYQNARYFTFWVGAGTSFNLSTLTEPTITPNTTQWNAFAPALDYSG 420 ****::**.*.:.. ****:*******.* ::*** :: ****...:* ******:*** FAdV-8-ON-NP1 APAFVYDANPVFTIYFQPNTGRVDTYLPVLTGDWKTSSTYNPGTITLVVRNATIQLQSQS 479 FAdV-8-ON-NP2 APAFVYDGNAVCTIYFQPNPGRVDTYLPVLTGDWKTSSTYNPGTITLVVRNATIQLQSQS 479 FAdV-8-ON-P1 APAFVYDANPVFTIYFQPNTGRLETYLPVLTDDWKTS-TYNPGTITLCVRTVRVQLRSQG 478 FAdV-8-ON-P2 APPFIYDASSVVTIYFEPTSGRLESYLPVLTDNWSQT--YNPGTVTLCVKTVRVQLRSQG 478 **.*:**...* ****:*..**:::******.:*. : *****:** *:.. :**:**. FAdV-8-ON-NP1 TFTTSVCYNFRCQNSGIFNNNATSGTLTLGPIFYSCPALSTADVS 524 FAdV-8-ON-NP2 TFTTSVCYNFRCQNSGIFNNNATSGTLTLGPIFYSCPALSTADVS 524 FAdV-8-ON-P1 TFNMLVCYNFRCQNAGIFNNNATAGTMTLGPIFFSCPALSTANAP 523 FAdV-8-ON-P2 TFSTLVCYNFRCQNTGIFNSNATAGTMTLGLIFFSCPALSTANAP 523 **. *********:****.***:**:*** **:********:..
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Figure 5.4. Supplementary data. Amino acid alignment of FAdV-11 fibers derived from flocks with and without signs of IBH. FAdV-11-ON-NP1 MAKSTPFAFSMGQHSSRKRPADSENTQNASKVAKTQTSATRAGVDGNDDLNLVYPFWLQN 60 FAdV-11-ON-NP2 MAKSTPFAFSMGQHSSRKRPADSENTQNASKVAKTQTSATRAGVDGNDDLNLVYPFWLQN 60 FAdV-11-ON-P2 MAKSTPFAFSMGQHSSRKRPADSENTQNASKVAKTQTSATRAGVDGNDDLNLVYPFWLQN 60 FAdV-11-ON-P1 MAKSTPFAFSMGQHSSRKRPADSENTQNASKVRQNADFCHARRCRRKYDLNLVYPFWLQN 60 ******************************** :. . : ************ FAdV-11-ON-NP1 STSGGGGGGSGGNPSLNPPFIDPNGPLYVQNSLLYVKTTAPIEVENKSLALAYDSSLDVD 120 FAdV-11-ON-NP2 STSGGGGGGSGGNPSLNPPFIDPNGPLYVQNSLLYVKTTAPIEVENKSLALAYDSSLDVD 120 FAdV-11-ON-P2 STSGGGGGGSGGNPSLNPPFIDPNGPLYVQNSLLYVKTTAPIEVENKSLALAYDSSLDVD 120 FAdV-11-ON-P1 STSGGGGGGSGGNPSLNPPFIDPNGPLYVQNSLLYVKTTAPIEVENKSLALAYDSSLDVD 120 ************************************************************ FAdV-11-ON-NP1 AQNQLQVKVDAEGPIRISPDGLDIAVDPSTLEVDDEWELTVKLDPAGPISSSSAGINIRV 180 FAdV-11-ON-NP2 AQNQLQVKVDAEGPIRISPDGLDIAVDPSTLEVDDEWELTVKLDPAGPISSSSAGINIRV 180 FAdV-11-ON-P2 AQNQLQVKVDAEGPIRISPDGLDIAVDPSTLEVDDEWELTVKLDPAGPISSSSAGINIRV 180 FAdV-11-ON-P1 AQNQLQVKVDAEGPIRISPDGLDIAVDPSTLEVDDEWELTVKLDPAGPISSSSAGINIRV 180 ************************************************************ FAdV-11-ON-NP1 DDTLLIEDDDTAQVKELGVHLNPNGPITADQDGLDLEVDPQTLTVTTSGATGGVLGVLLK 240 FAdV-11-ON-NP2 DDTLLIEDDDTAQVKELGVHLNPNGPITADQDGLDLEVDPQTLTVTTSGATGGVLGVLLK 240 FAdV-11-ON-P2 DDTLLIEDDDTAQVKELGVHLNPNGPITADQDGLDLEVDPQTLTVTTSGATGGVLGVLLK 240 FAdV-11-ON-P1 DDTLLIEDDDTAQVKELGVHLNPNGPITADQDGLDLEVDPQTLTVTTSGATGGVLGVLLK 240 ************************************************************ FAdV-11-ON-NP1 PSGGLQTSIQGIGVAVADTLTISSNTGTVEVKTDPNGSIGSSSNGIAVVTDPAGPLTTSS 300 FAdV-11-ON-NP2 PSGGLQTSIQGIGVAVADTLTISSNTGTVEVKTDPNGSIGSSSNGIAVVTDPAGPLTTSS 300 FAdV-11-ON-P2 PSGGLQTSIQGIGVAVADTLTISSNTGTVEVKTDPNGSIGSSSNGIAVVTDPAGPLTTSS 300 FAdV-11-ON-P1 PSGGLQTSIQGIGVAVADTLTISSNTGTVEVKTDPNGSIGSSSNGIAVVTDPAGPLTTSS 300 ************************************************************ FAdV-11-ON-NP1 NGLSLKLTPNGSIQSSSTGLSVQTDPAGPITSGANGLSLSYDTSDFTVSQGMLSIIRNPS 360 FAdV-11-ON-NP2 NGLSLKLTPNGSIQSSSTGLSVQTDPAGPITSGANGLSLSYDTSDFTVSQGMLSIIRNPS 360 FAdV-11-ON-P2 NGLSLKLTPNGSIQSSSTGLSVQTDPAGPITSGANGLSLSYDTSDFTVSQGMLSIIRNPS 360 FAdV-11-ON-P1 NGLSLKLTPNGSIQSSSTGLSVQTDPAGPITSGANGLSLSYDTSDFTVSQGMLSIIRNPS 360 ************************************************************ FAdV-11-ON-NP1 AYPDAYLESGTNLLNNYTAYAENSSNYKFNCAYFLQSWYSNGLVTSSLYLKINRDNLTSL 420 FAdV-11-ON-NP2 AYPDAYLESGTNLLNNYTAYAENSSNYKFNCAYFLQSWYSNGLVTSSLYLKINRDNLTSL 420 FAdV-11-ON-P2 AYPDAYLESGTNLLNNYTAYAENSSNYKFNCAYFLQSWYSNGLVTSSLYLKINRDNLTSL 420 FAdV-11-ON-P1 AYPDAYLESGTNLLNNYTAYAENSSNYKFNCAYFLQSWYSNGLVTSSLYLKINRDNLTSL 420 ************************************************************ FAdV-11-ON-NP1 PSGQLSENAKYFTFWVPTYESMNLSNVATPTITPSSVPWGAFLPAQNCTSNPAFKYYLTQ 480 FAdV-11-ON-NP2 PSGQLSENAKYFTFWVPTYESMNLSNVATPTITPSSVPWGAFLPAQNCTSNPAFKYYLTQ 480 FAdV-11-ON-P2 PSGQLSENAKYFTFWVPTYESMNLSNVATPTITPSSVPWGAFLPAQNCTSNPAFKYYLTQ 480 FAdV-11-ON-P1 PSGQLSENAKYFTFWVPTYESMNLSNVATPTITPSSVPWGAFLPAQNCTSNPAFKYYLTQ 480 ************************************************************ FAdV-11-ON-NP1 PPSIYFEPESGSVQTFQPVLTGDWDTNTYNPGTVQVCILPQTVVGGQSTFVNMTCYNFRC 540 FAdV-11-ON-NP2 PPSIYFEPESGSVQTFQPVLTGDWDTNTYNPGTVQVCILPQTVVGGQSTFVNMTCYNFRC 540 FAdV-11-ON-P2 PPSIYFEPESGSVQTFQPVLTGDWDTNTYNPGTVQVCILPQTVVGGQSTFVNMTCYNFRC 540 FAdV-11-ON-P1 PPSIYFEPESGSVQTFQPVLTGDWDTNTYNPGTVQVCILPQTVVGGQSTFVNMTCYNFRC 540 ************************************************************ FAdV-11-ON-NP1 QNPGIFKVAASSGTFTIGPIFYSCPTNKLTQP 572 FAdV-11-ON-NP2 QNPGIFKVAASSGTFTIGPIFYSCPTNKLTQP 572 FAdV-11-ON-P2 QNPGIFKVAASSGTFTIGPIFYSCPTNKLTQP 572 FAdV-11-ON-P1 QNPGIFKVAASSGTFTIGPIFYSCPTNKLTQP 572 ********************************
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GENERAL DISCUSSION
Fowl adenoviruses are common infectious agents of poultry and often of low
virulence. The widespread occurrence of antibodies and the isolation of FAdVs from
clinically healthy chickens argue against a role of FAdVs in primary poultry disease
(Yates et al., 1977; Okuda et al., 2006; Ojkic et al., 2008a). Common to all virulent
FAdVs is infection of hepatocytes which results in a clinical manifestation known as IBH
occurring mainly in broilers. FAdVs are shed in feces and usually spread horizontally
through contaminated food, water, and litter (McFerran and Smyth, 2000). Evidence of
vertical transmission has also been reported (Grgic et al., 2006). From the available
evidence, it seems that most strains of FAdVs follow the same pattern of infection.
Following initial multiplication, viraemia occurs and results in virus spread to virtually
all organs. The main sites of virus replication are the respiratory and alimentary systems
(Saiffudin and Wilks, 1991). In our study, two Canadian FAdV isolates were evaluated:
FAdV-8 was isolated from an IBH outbreak that occurred in an Ontario broiler farm, and
FAdV-4 was isolated from a broiler breeder flock showing no clinical signs of IBH or
IBH/HHS. Pathogenicity was determined for both isolates on the basis of clinical signs
and pathological and histological examination. Additionally, viral genome copy numbers
in the bursa of Fabricius, cecal tonsil, and liver were evaluated following infection, and
virus titer in feces and antibody response were also determined.
According to Ojkic and Nagy (2003), replication and distribution of FAdVs in
chicken tissues is markedly influenced by the route of virus inoculation. Therefore, we
compared im and the more natural oral routes of inoculation. Despite a lack of clinical
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signs and pathological changes, both FAdV-8 and FAdV-4 were found in tissues and
cloacal swabs of all birds. For both FAdV-8 and FAdV-4 and in both the orally and
intramuscularly inoculated groups, the highest numbers of viral genome copies were
found in the cecal tonsil. Virus was isolated from cloacal swabs up to 28 days post
infection for both viruses, which is believed to be the longest recorded shedding period
for fowl adenoviruses. Both FAdV-8 and FAdV-4 were sufficiently immunogenic to
induce a strong antibody response.
The pathogenicity data indicated that under the conditions of this study, neither
isolate was pathogenic. Thus, the effects of FAdV infection on the transcription of a
number of avian cytokines were investigated. Cytokines are important mediators of
inflammation and regulators of the immune response. The first defense against viral
infection is an inflammatory response, with simultaneous release of inflammatory
cytokines. However, the role of interleukins in the pathogenicity of and immune response
to FAdVs is unknown. This study explored the ability of fowl adenoviruses to subvert the
host cells’ secretion of cytokines in response to infection as an important viral
mechanism for immune evasion during infection. In chicken experiments, IFN-γ, IL-10,
IL-18, and IL-8 gene expression were evaluated following FAdV-8 and FAdV-4
infection. Cytokine gene expression was examined in the liver, spleen, and cecal tonsils.
Results showed that IFN-γ, IL-18, IL-10 and IL-8 were activated early in FAdV
infection. The studied cytokines represent chicken Th1 and regulatory cytokines and all
have previously been shown to be involved in immunity to viral infections. IFN-γ,
considered an essential cytokine in host defense against a variety of pathogens, inhibits
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MDV replication (Xing and Schat, 2000), reduces replication of coccidian parasites
(Lillehoj and Choi, 1998), and inhibits src oncogene-induced tumor growth (Plachy et al.,
1999). In this study, the IFN-γ gene was induced to a significant degree following both
FAdV-8 and FAdV-4 infection. Another Th1 cytokine, IL-18, was originally identified as
a factor promoting IFN-γ production by T and NK cells when costimulated by IL-12
(Okamura et al., 1995). This proinflammatory cytokine (Gobel et al., 2003) is involved in
activation of mammalian neutrophils and NK cells (Okamura et al., 1995). Following
FAdV-8 infection, expression of the IL-18 gene was significantly higher in spleen and
liver in infected chickens than in mock-infected chickens. Enhanced expression of IL-18
in liver and cecal tonsil was detected in FAdV-4 infection. However, there was no
significant difference between infected and mock-infected chickens in terms of IL-18
gene expression. The increase in expression of IL-18 is probably an indicator of induction
of inflammation and an immune response against the virus. Furthermore, IL-10 was up-
regulated following FAdV-8 and FAdV-4 infections, indicating an immune evasive
mechanism used by the virus to skew the immune response against FAdVs, supporting
persistence of the virus. Simultaneous expression of IFN-γ and the IL-10 gene also has
been reported by Johensen et al, (2002) and Parvizi et al (2009). It is possible that
enhanced expression of IL-10 is induced by the chicken immune system to modulate the
immune response and inflammation caused by the virus. The increased expression of
mRNA of the IL-8 gene in the liver recorded after FAdV-4 infection could be a reactive
response of the host to reduce viral load. In contrast, IL-8 mRNA was significantly down-
regulated in the liver and spleen following FAdV-8 infection. Inhibition of IL-8 mRNA
expression by mammalian adenovirus E3 genes has been reported by Lesokhin et al,
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(2002). However, FAdV-8 lacks the mastadenoviral E3 region (Grgic et al., 2011). Thus,
some parts of the FAdV-8 genome probably share a function similar to the
mastadenoviral E3 region, despite the lack of obvious sequence homology. This study
provides useful information about important immunological mediators involved in
immunity against FAdVs and virus evasion that could lead to an effective vaccine against
IBH or IBH/HHS.
In the family Adenoviridae, viruses belonging to the genus Mastadenovirus have
been extensively investigated, and their genomic organizations have been well
characterized. Although aviadenoviruses have been less studied, recently Grgic et al,
(2011) and Griffin and Nagy (2011) reported the complete sequences of two FAdV
serotypes (FAdV-8 and FAdV-4). In addition, the sequence analysis of the left and right
end regions of FAdVs representing each species group has been also determined
(Corredor et al., 2006; Corredor et al., 2008).
Here, for the first time, a comparative analysis of the full genome sequence of an
FAdV-8 isolate of the species Fowl adenovirus E has been described. The FAdV-8
genome is 251 nt longer than that of the FAdV-1 genome and 1008 nt shorter than that of
the FAdV-9 genome (Chiocca et al., 1996; Ojkic and Nagy, 2000). Several important
features of FAdV-8 contribute to this size difference. The nucleotide and amino acid
sequence analyses revealed a lack of mastadenoviral early regions E1, E3, and E4 and the
sequences of proteins V and IX. Lack of early regions has been also reported for FAdV-1
and FAdV-9 genomes (Chiocca et al., 1996; Ojkic and Nagy, 2000). Tandem repeats,
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short repeated regions located at the right end of the FAdV-9 genome with unknown
function and non-essential for genome replication, have been identified within ORF19 in
the FAdV-8 genome. However, tandem repeats of FAdV-8 are much shorter than those of
FAdV-9. Virus-associated RNA found in mammalian adenoviruses is 150 to 170 nt long
and transcribed rightward (Ma and Mathews, 1993). Human AdVs contain one or two
VA RNA genes located between the pTP and 52k ORFs. In contrast, the VA RNA of
FAdV-1 is shorter, comprising only 100 nt and transcribed leftward (Larsson et al.,
1986). Although the VA RNA in the EDS virus is similar in size to that in FAdV-1,
comprising 91 nt, it is transcribed rightward as in mammalian adenoviruses (Hess et al.,
1997). Sequence analysis showed that the VA RNA gene was not identified in the FAdV-
8 genome. The lack of a VA RNA gene was also reported for FAdV-9, FAdV-4, and
OAdV287 (Ojkic and Nagy, 2000, Griffin and Nagy, 2011, Venktesh et al., 1998). In
contrast to FAdV-1, which has two fiber genes (Chiocca et al., 1996), only one fiber gene
was identified in the corresponding region of the FAdV-8 and FAdV-9 genomes (Grgic et
al., 2011; Ojkic and Nagy, 2000). The fiber gene has been implicated in pathogenicity of
HAVs (Mei and Wadell, 1995) and variation in virulence of FAdVs (Pallister et al.,
1996). Although extensive research with a focus on structure, sequence analysis, and
pathogenicity (El Bakkouri et al., 2008; Grgic et al., 2011; Griffin and Nagy, 2011) was
performed, determinants of virulence for FAdVs are still unknown. I hypothesized that
sequence variations of the fiber gene contribute to the virulence of FAdVs. Therefore, the
nucleotide and amino acid structure of the fiber gene of eight fowl adenoviruses
(pathogenic and non-pathogenic) representing species groups D (FAdV-11) and E
(FAdV-8) were analyzed. The FAdV-11 and FAdV-8 fiber genes encoded 572 aa and 522
146
to 525 aa, respectively, and like all other adenovirus fibers, they are organized into three
regions, tail, shaft, and head. The tail region of both serotypes 8 and 11 contained a
poly(G) stretch identified previously in the tail of the long fiber of FAdV-1 (Hess et al.,
1995), while the same stretch is absent in the FAdV-10 fiber gene (Sheppard et al.,
1998). It was shown for mammalian AdVs (Green et al., 1983; van Raaij et al., 1999)
that the shaft region contains repetitive elements. Correspondingly, the shaft region of
FAdV-11 contains 20 pseudorepeates, while FAdV-8 contains 17 pseudorepeates, each
repetition comprising 18 and 16 aa on average, respectively. Moreover, mastadenovirus
conserved amino acid sequence (KLGXGLXFD/N) is absent from both FAdV-8 and -11
fibers and has not been reported for FAdV-1 and FAdV-10 (Hess et al., 1995; Sheppard
et al., 1998). Well-conserved TLWT sequence in the first β-strand of the head (Xia et al.,
1994), also was not evident in either FAdV-8 or FAdV-11 fibers. From these results it
may be concluded, that virulence might not be associated with the sequence of the fiber
gene alone. Experimental infection would be necessary to estimate the influence of the
adenoviral fiber gene on pathogenesis. In conclusion, this is the first study aimed at
determining cytokine gene expression patterns associated with FAdV infection in vitro
and in vivo.
Additionally, on the basis of a complete genome sequence analysis of FAdV-8, it
was concluded that FAdV-1 of the species Fowl adenovirus A as the current type species
is not the best representative of the genus Aviadenovirus, and that FAdV-8 in the species
Fowl adenovirus E would be more suitable for that designation.
147
Data generated from this work provide the basis for future studies related to
cytokine gene expression patterns associated with FAdV infection and to virulence
factors in pathogenic FAdVs.
148
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