A Review on Inclusion Body Hepatitis (IBH): An Emerging Problem in Sri Lanka

: Viruses belong to the Family Adenoviridae can infect a wide range of vertebrates. These viruses are classified into different genera based on phylogenetic distance, genome organization, and host range. From those genera, Aviadenovirus only infect birds and are serologically different from other adenovirus genera . Fowl Adenoviruses (FAdVs) fall under the Aviadenovirus genus and have a considerably large genome. The FAdV genome comprises non-segmented, linear double-stranded DNA (dsDNA) of 43-46 kbp. The capsid of FAdVs is composed of hexons, penton bases, and fiber proteins, which are vital for cellular interaction and immune responses. Inclusion Body Hepatitis (IBH) is a significant disease caused by FAdVs, impacting broiler chickens primarily. Inclusion Body Hepatitis results in sudden mortality, poor growth, and other clinical signs, contributing to economic losses in the poultry industry, and is transmitted horizontally through contaminated excretions and vertically via embryonated eggs from infected parent flocks. The disease pathogenesis involves replication in the alimentary and respiratory tract epithelium, leading to viremia and dissemination to organs. Gross pathological findings mainly include swollen liver with necrotic foci and multifocal petechial hemorrhages. Histopathologically, degeneration, necrosis, and the presence of intranuclear/intracytoplasmic, basophilic/eosinophilic inclusion bodies are observed mainly in hepatocytes. Diagnosis of IBH involves detecting intranuclear inclusion bodies in hepatocytes, virus isolation, and molecular techniques like PCR for serotyping. Control strategies include strict biosecurity measures, continuous disease surveillance, breeder monitoring, and vaccination. Inactivated and live vaccines are commonly used, with autogenous inactivated vaccines prepared from prevalent serotypes for prevention in endemic regions. Since 2021, IBH outbreaks have been reported in Sri Lanka. The objective of this review is to provide a quick overview of the members and viral structure of the Family Adenoviridae as well as an in-depth review of IBH, one of the main diseases caused by FAdV in poultry.


INTRODUCTION
Inclusion body hepatitis (IBH) is caused by Fowl adenoviruses (FAdVs), classified under Family Adenoviridae.Family Adenoviridae contain viruses which are non-enveloped and have icosahedral capsid with one linear double-stranded deoxyribonucleic acid (dsDNA) genome of 25-48 kbp (ICTV;2021).The members of the Family adenoviridae can infect vast range of vertebrate, from fish to humans (Benkő et al., 2022).

Family Adenoviridae Taxonomy
Based on the phylogenetic distance, genome organization, and host range, Family Adenoviridae can be divided into six distinct genera: Mastadenovirus, Atadenovirus, Aviadenovirus, Ichtadenovirus, Testadenovirus and Siadenovirus (ICTV; 2021).From those genera, Aviadenovirus only infect birds and are serologically different from other adenovirus genera.Also they have a considerably large genome (ICTV; 2021).FAdVs belonged to the genus Aviadenovirus.

Genus Aviadenovirus Classification
According to the International Committee on Taxonomy of Viruses, 2021, genus Aviadenovirus has sixteen species namely Aviadenovirus leucophthalmi, Falcon aviadenovirus A, Duck aviadenovirus B, Fowl aviadenovirus (FAdV) A, Fowl aviadenovirus B, Fowl aviadenovirus C, Fowl aviadenovirus D, Fowl aviadenovirus E, Goose aviadenovirus A, Pigeon aviadenovirus A, Pigeon aviadenovirus B, Psittacine aviadenovirus B, Psittacine aviadenovirus C, Turkey aviadenovirus B, Turkey aviadenovirus C, and Turkey aviadenovirus D. There is another classification of genus Aviadenovirus according to the biological diversity as previously described by Hess, 2000.In this classification genus Aviadenovirus was divided into three groups and it is demonstrated in table 1.

Structural Elements of the Family Adenoviridae Capsid and the Viral Core
The icosahedral capsid consists of 240 non-vertex capsomers (hexons) of 8-10 nm in diameter, and 12 vertex capsomers (penton bases) with one or two protruding fiber (Van Raaij et al., 1999).As Benkö & Harrach, 2003 said, family Adenoviridae exhibits a common structure, but genus Aviadenovirus consisted of two fibers projected from the penton base (Adair & Fitzgerald, 2008;Gelderblom & Maichle-Lauppe, 1982).Thus, hexons, penton base and the protruding fiber/s are considered as three major structural proteins of the icosahedral adenoviral capsid (Sohaimi & Hair-Bejo, 2021;Russell, 2009;Crawford-Miksza & Schnurr, 1996).Capsid minor components are polypeptide (p) IIIa, p VI, p VIII and p IX (Vellinga et al., 2005).That p IIIa, p VI, p VIII are located on the inner capsid surface, while p IX is located on the outer surface of the capsid (Gallardo et al., 2021).A schematic diagram of the adenovirus capsid major and minor proteins is given in figure 1.The main functions of capsid major and minor proteins of family Adenoviridae are given in table 2.
Hexon is a homo-trimer (3 polypeptide sub units) (Athappily et al., 1994) that gives a pseudohexagon shape and contributes a large proportion of the capsid (Sohaimi & Hair-Bejo, 2021).Hexon protein has conserved, internally located, pedestal regions (P1 and P2) and variable loops (L1, L2 and L4 located outer surface of the capsomer and L3 is buried inside the capsomer).L1, L2 and L4 are considerd as type-specific epitopes while the L1 loop has the most variable region and it is the longest loop.Therefore the gene contributes for the variable region of L1 loop is used for serotyping of FAdVs (Sohaimi & Hair-Bejo, 2021;Crawford-Miksza & Schnurr, 1996).The fiber protein has N-terminal tail attached to the penton base, thin shaft which the length varies depending on serotype and carboxyl-terminal head which is having receptor binding site (Mathis et al., 2005).
The viral core has the viral genome together with six protein structural elements, five of which are connected to the double-stranded DNA genome [p V, p VII, Mu, IVa2, and the terminal protein (TP)], while the sixth is the 23K virion protease, which is crucial for the virion's construction (Russell, 2009).A schematic diagram of Adenovirus core structure is given in figure 2. The main functions of viral core proteins of family Adenoviridae are given in table 3.

Family Adenoviridae Genome
An inverted terminal repeat (ITR) with sizes ranging from 36 to over 200 kbp characterized the genome of the Family Adenoviridae.Also, the 5′ ends are connected to a terminal protein (TP).16 distinct genus-common genes have been identified in the Family Adenoviridae genome.Their main roles are in DNA replication, DNA encapsidation, virion generation and formation of virion structure.Towards the ends of the genome is where the majority of genusspecific genes are found (Davison et al., 2003).Despite the substantial sequence variability amongst the different species, the Adenoviridae genome is organized in a unique and highly conserved way (Tibbetts, 1977).
Main four gene transcription units have been identified as S. A. S. Indunika et al.   (Kajon et al., 2019;Hall et al., 2010;Berk, 2005).A schematic diagram of adenovirus genome organization is given in figure 3.

Species FAdVs -Genome
FAdVs have non-segmented, linear dsDNA genomes which is considerably larger than the other viruses in the family Adenoviridae (ICTV, 2021;Park et al., 2012).The central part of the genome is well conserved throughout the family, while the two ends show a wide variation in length and content making FAdVs genetically different (Park et al., 2012).
By looking at the table 5 where the genome size is demonstrated, FAdVs have large genome ranging from 43 -46 kbp.

Inclusion Body Hepatitis (IBH)
Inclusion body hepatitis is an acute disease, mainly affecting 3-7 weeks old broiler chickens (Winterfield et al., 1973).It was first reported in 1963 in the USA (Panigrahi et al., 2016).Even though IBH is affecting 3-7 weeks old chickens, cases has been reported of affecting 7 days old chicks and in 20 weeks old chickens (Panigrahi et al., 2016).

Emergence and Distribution of IBH
Inclusion body hepatitis was first reported by Helmboldt and Frazier in the USA in 1963.It was initially described as a necrotizing hepatitis in 7-week-old chickens associated with Cowdry type "A" intranuclear inclusion bodies in hepatocytes.Subsequently, it was shown that the disease was associated with infection by an adenovirus (Fadly & Winterfield, 1973).IBH has been reported in broiler flocks in several other countries/regions of the world, including Canada (Howell et al., 1970), UK (Pettit & Carlson, 1972), Australia (Wells & Harrigan, 1974), and New Zealand (Christensen & Saifuddin, 1989).Although IBH has been reported as clinical outbreaks in many countries, there is still a scarcity of published information on the occurrence of this disease complex in Sri Lanka.

Economic Impact of IBH
Poultry is one of the high income generating industry and become more popular globally.There is a high demand for poultry meat and eggs.When considering the time between 1961 and 2020 poultry meat production increased from 9 to 133 million tones whereas egg production increased from 15 to 93 million tones (FAO, 2023).Also, poultry meat represents about 40 percent of world meat production in the year 2020.Chicken is the most abundant meat type accounting 90 percent of global poultry meat and 94 percent of world poultry population is contributed by chicken in 2020 (FAO, 2023).In Sri Lanka 64 percent of the Gross Domestic Production (GDP) in 2020 from the livestock sector was from poultry (DAPH, 2021) According to this global statistical data, disease condition such as IBH can be drastically reducing poultry production.This effect aggravated by the factors such as worldwide distribution of FAdVs.Recent studies found out that IBH outbreaks become more epidemic outbreaks than sporadic outbreaks (El-Shall et al., 2022).Studies carried out in North America have shown heavy mortalities, weight loss and high condemnation at slaughter houses associated with IBH in broilers (Abghour et al., 2019;Gomis et al., 2006).

Transmission of IBH
Inclusion body hepatitis can be transmitted horizontally and vertically (Panigrahi et al., 2016).Vertically transmitted by viremic parent flocks to the chicks through embryonated eggs and clinical signs can be seen in the chicks during their first weeks of life, especially if they are immunosuppressed (McFerran & Adair, 1977).Horizontal transmission occurs by excretions (mainly by contaminated feces) of the birds and contaminated fomites, personnel, and transport (McFerran & Adair, 2003).The ability of both vertical and horizontal transmission has lead FAdV to become a worldwide pathogen (Adair & Fitzgerald, 2008).

Morbidity and Mortality of IBH
The morbidity of IBH is low (around 10%) (Gomis et al., 2006).Mortality usually ranges from 5% to 10% but can reach 30%.Natural outbreaks of IBH are characterized by a sudden onset of mortality which peaks within 3-4 days and return to normal by 5-6 days (Abghour et al., 2019).High mortality can be seen in birds less than 3 weeks old (Panigrahi et al., 2016).Some studies concluded that mortality in IBH can be 10% to 50% depending on virus strain and host susceptibility (Matos et al., 2016).
Depending on the pathogenicity of the virus, immune status of the chicks and concurrent secondary infections, mortality can reach up to 80%.

Gross Pathology of IBH
A field study revealed severe hepatomegaly, splenomegaly, renomegaly and icterus as gross lesions on the necropsy (Mariappan et al., 2018).A study in broilers in Kermanshah province, Iran, obsverved that the liver was the primarily afected organ having hepatomegaly, pale yellowish liver with necrotic foci and multi focal petechial hemorrages (Rahimi & Haghighi, 2015).The same observation has been done by Reece et al., 1986.The affected liver can be friable (Cizmecigil et al., 2020).Multifocal pale necrotic areas on the surface of the spleen (Mariappan et al., 2018) and diffuse multi focal hemorrhages on spleen have been observed (Cizmecigil et al., 2020).
Petechial hemorrhages may be present in skeletal muscles and severe mucosal congestion with excess catarrhal exudate in the lumen of the intestines can be seen in a necropsy (Wilson et al., 2010).Pancreas and kidneys become swollen and pale (Cizmecigil et al., 2020).In some cases, tubular pattern of the kidneys has become prominent grossly (Wilson et al., 2010).

Histopathology of IBH
Large basophilic intra-nuclear inclusion bodies in hepatocytes and acinar cells of pancreas are prominent in severely affected birds accompanied with degeneration and necrosis (Matos et al., 2016).Degenerating hepatocytes with one or more large vacuoles in the cytoplasm and varying degrees of pyknosis, karyorrhexis and karyolysis and some hepatocytes with lost nuclei leaving a ghost cell appearance can be detected (Panigrahi et al., 2016).In some cases basophilic or eosinophilic intranuclear inclusions in the degenerating hepatocytes can be seen (Alvarado et al., 2007) which are called large Cowdry Type A intranuclear inclusion bodies.Multifocal lymphocyte infiltration in liver and pancreas has been reported in some studies (Matos et al. 2016).Congestion and focal hemorrhage are observed in kidneys.In addition, myocardial edema in the heart with degeneration, necrosis, and mild mononuclear cell infiltration can be observed.

Diagnosis of IBH
Laboratory diagnosis can be done histologically by detection of intra-nuclear inclusion bodies in hepatocytes (Panigrahi et al., 2016;Wilson et al., 2010).Virus identification by electron microscopy (McFerran, 1998) and detection of virus particles or the antigen by immunofluorescence assay is commonly used for the diagnosis of IBH (Panigrahi et al., 2016).But electron microscopy alone cannot provide group-specific information and subgroup-specific information of the virus (Joubert, 2000).As Hess, 2000 stated, clinical history and necropsy findings are useful diagnostic tools.Serological methods (such as serum neutralization) have little significance of identifying FAdV isolates (Benkő et al., 2005).
FAdVs are commonly isolated in chicken kidney (CK) or chicken embryo liver (CEL) cells.Embryonated eggs have also been reported as a sensitive medium for replication of virus, if injected via the yolk sac route (Cowen, 1988).
Molecular diagnosis of FAdVs has become a common practice recently.The loop 1 (L 1) region of the hexon gene is used for the serotyping of FAdVs due to the high variability of L1 loop among different serotypes (Meulemans et al., 2004) and high vulnerability for mutations which having the major neutralizing epitope.
Polymerase Chain Reaction (PCR) is used for the identification of the hexon gene.Different primer sets are available for amplification of the various regions of hexon gene (can use a specific primer set for L1 region of the hexon gene) (Steer et al., 2008).Obtained PCR products can be used for nucleotide sequencing or restriction enzyme digestion for categorization of field isolates into species and serotypes (Meulemans et al., 2001).The combination of PCR and high-resolution melting curve analysis has also provided an accurate and rapid genotyping technique for the identification of FAdV serotypes (Grafl et al., 2013).

Differential Diagnosis of IBH
Diseases with hemorrhagic diathesis such as vitamin K S. A. S. Indunika et al. deficiency (Chaves Hernández, 2014), mycotoxicosis, intoxication by sulfonamides and infectious bursal disease (Hoffmann et al., 1975) can be considered as differential diagnosis of IBH

IBH Control
According to the available literature, IBH infections were recorded throughout the year but more prominent in rainy season.Therefore, the control of IBH is very important.Continuous disease surveillance, breeder monitoring and vaccination are the most effective prevention and control strategies (Bertran et al., 2021).
Good biosecurity measures such as cleaning and disinfecting poultry houses and equipment, foot bath constructing, visitor restriction, record keeping and other management practices and training workers are primary control methods (El-Shall et al., 2022).Even though, some studies showed that very strict biosecurity measures of parent flocks increase the occurrence of FAdV-related diseases.The reason for this incident that is suggested by the study is, not transmitting maternal antibodies to progeny due to lack of natural immunity of parent flocks (Schachner et al., 2018).
Detection and identification of FAdVs strains circulating in a particular country/area is very important in monitoring disease outbreaks and implementation of vaccination programs (Schachner et al., 2018).In FAdVs endemic countries, inactivated and live vaccines are used (Monis et al., 2005).Broiler-breeders are vaccinated with autogenous vaccines to develop immunity against IBH in progeny through maternal antibody (Gomis et al., 2006;Alvarado et al., 2007).Even though the autogenous vaccines are used, sporadic outbreaks of IBH occur in the vaccinated chicks (Ojkic et al., 2008)

IBH Vaccines
In recent times, IBH outbreaks have resulted in higher mortality in birds compared to previous milder outbreaks, leads to severe economic losses to the poultry sector.
As a result, it is possible that the virulence of virus has changed, causing high mortality (Saifuddin & Wilks, 1990).To overcome IBH, many countries use both live and inactivated vaccines.FAdV serotypes 4 and 8 are the most commonly used in the preparation of commercial vaccines (Saifuddin & Wilks, 1990).Moreover, autogenous inactivated vaccines prepared from the prevalent serotype of FAdV are recommended for disease prevention and control in endemic areas.Autogenous inactivated vaccines were used by primary breeders with strict biosecurity practices to prevent vertical transmission and ensure the transfer of maternal immunity from breeding flocks to their progeny (Saifuddin & Wilks, 1990).
In the United States, where biosecurity is strictly enforced, autogenous inactivated vaccines are commonly used in primary breeder flocks.By immunizing a grandparent stock at 10 and 17 weeks of age with an inactivated vaccine containing isolates of species FAdV-D and FAdV-E, full protection against IBH was achieved in breeders up to the age of 50 weeks (Alvarado et al., 2007).
In Australia, breeders were given a live vaccine (FAdV-8b) 1-3 times by drinking water between the ages of 9 and 18 weeks; however, IBH outbreaks in commercial broilers continue.A heterologous strain was found rarely in sick birds alongside a FAdV similar to the vaccine, indicating a lack of cross-protection (Steer et al., 2015).An inactivated oil-emulsion FAdV-4 vaccine, on the other hand, produced high levels of protection against various serotypes in both vaccinated/challenged SPF chickens and broilers descended from vaccinated breeders (Kim et al., 2014).

IBH Infection in Sri Lanka
Even though the scarcity of published data on IBH condition in Sri Lanka, it is believed that the disease prevalence is high in field condition.During July 2019 to March 2020, sudden clinical outbreak was reported in 13 commercial broiler farms and one layer farm in Kurunegala, Puttalam and Gampaha districts which was suggestive of IBH (Perera et al., 2021).According to the above mentioned cases, sudden onset of mortality has been reported ranging from 1% to 18%.Varying degrees of multifocal hepatocellular degeneration and necrosis with infiltration of lymphocytes, macrophages and some heterophils were the histological finding of samples submitted from all the 14 farms (n=112/123).In birds of 12 farms, mostly basophilic intra-nuclear inclusion bodies in the hepatocytes have been detected with occasional eosinophilic intranuclear inclusions surrounded by a halo (53/123).The diagnosis as IBH has been done with clinical signs, post mortem findings and histopathological findings (Perera et al., 2021).This was the first reported IBH outbreak in Sri Lanka as mentioned by Perera et al., 2021.

CONCLUSION AND FUTURE PERSPECTIVES
Inclusion body hepatitis is currently classified as an emerging poultry disease.However, the pathogenicity of the majority of isolates is still unclear.Co-infections with other immunosuppressive viruses such as infectious bursal disease virus, avian leucosis virus and chicken anemia virus significantly worsen the severity of infection in the field.Clinical diagnosis, molecular detection of virus using PCR and histopathology are the most effective methods for identifying IBH in birds.To control the disease, breeders must implement strict biosecurity procedures, appropriate management methods and immunization programs.As a result, the continued growth of the chicken industry is dependent on expanding research into diseases caused by adenoviruses.

ACKNOWLEDGEMENT
Authors acknowledge the financial assistance given by the University of Peradeniya under its University Research Grant Scheme (URG/2021/43/V and URG/2021/41/V).

FowlFigure 1 :
Figure 1: Adenovirus capsid structure.Left: 3D structure of the viroin; Yellow -penton bases, dark blue -fiber protein trimers, shaded triangle -one facet.Right: schematic diagram of one triangular facet with major and minor proteins.Source: San Martin, 2012 (reprinted with permission).

Figure 2 :
Figure 2: Schematic diagram of Adenovirus core structure.Source: Gallardo et al., 2021 (reprinted with permission).Table 2: Main functions of the Family Adenoviridae capsid major and minor proteins Protein Main function Hexon • Initiating cellular and innate immune responses (Russell, 2009) • Stabilizing the virion structure (Russell, 2009) Penton base • Stabilizing the virion structure (Russell, 2009) • Part of the capsid and sensitive to heat, trypsin, pH and ionic strength changes (Rexroad et al., 2006) Fiber • Tissue tropism (Russell, 2009) • The first component come into contact with the tissue (Russell, 2009) • Heamagglutinin (Pehler-Harrington et al., 2004) p IIIa • Initiating virus disassembly (Russell, 2009) p VI • Facilitate the entry of the virus into the host cell (Gallardo et al., 2021) • Aid the transport of newly translated hexon into the host cell nucleus and virus assembly (Wodrich et al., 2003) p VIII • Stabilizing hexons in the capsid (Gallardo et al., 2021) p IX• Provide thermo-stability of the capsid(Vellinga et al., 2005)

Table 1 :
Group differentiation and classification of genus Aviadenovirus

Table 3 :
Main functions of the Family Adenoviridae viral core proteins Viral core proteins Main function p V • Provide stability to the virion (Zhao & Tikoo, 2016) p VII • Facilitate the transport of virus genome into the host cell nucleus (Kulanayake & Tikoo, 2021) Mu • Involve in the condensation of virus ds DNA molecule inside the virion (Russell, 2009) IVa2 • Activates the main late promoter of the adenovirus (Mondesert et al., 1992) TP • linked to the terminal 5' of the genome segment and engaged in DNA replication (Hoeben & Uil, 2013) Protease • Breaking down of precursor proteins in the viral genome • (Mangel & San Martin, 2014) early region, intermediate region, major late region and virus associate RNAs as described in table 4

Table 4 :
Main gene transcription units of the family Adenoviridae genome and their functions

Table 5 :
Size of the genome of FAdVs in base pairs (bp)

Table 6 :
Common poultry diseases caused by different serotypes of FAdVs