Genetic diversity of fowl adenovirus serotype 8b isolated from cases of inclusion body hepatitis in commercial broiler chickens

Inclusion body hepatitis (IBH) is a devastating disease of chickens caused by Fowl adenovirus (FAdV) and molecular studies of hexon gene is important for classification of FAdV isolates, epidemiology of IBH infection and development of effective control strategy. The objective of the study was to molecularly characterize FAdV isolates obtained from the field outbreaks of IBH in broiler chicken from 2017 to 2019. Liver and gizzard samples named UPM1701, UPM1801, UPM1802, UPM1901 and UPM1902 were collected and processed for FAdV detection and characterization by PCR. Swollen, necrotic and haemorrhagic liver; erosion of koilin layer of gizzard and enlargement of the proventriculus were observed. The samples were positive for FAdV with 98% to 100% identical to serotype 8b reference strains based on NCBI Blast. Thirty-one nucleotide changes that produced 10 amino acid substitutions were observed in L1 loop region of UPM1701, UPM1901 and UPM1902 isolates. All isolates clustered together with FAdV 8b reference strains and shared common ancestor with UPM1137E2 and UPM04217. Gizzard erosion in FAdV 8b infection is not common and conciding with mutations indicate evolving novel pathogenicity. These mutations could have effect on the epidemiology of IBH and be useful in designing effective prevention and control strategy.


INTRODUCTION
Fowl adenovirus (FAdV) is classified into 5 molecular species designated A to E and 12 serotypes (Steer et al., 2011;Morshed et al., 2017) and has been reported to be endemic in various geographical regions worldwide where they cause high levels of economic losses among poultry species (Li et al., 2018;Schachner et al., 2018;Jordan et al., 2019). FAdV are nonenveloped, double stranded DNA virus comprised of three major structural proteins known as hexon, penton and fiber (Park et al., 2016). Antigenic and most of the virulence determinants for FAdV is located in mainly hexon gene (Park et al., 2016); although fiber proteins has become highly relevant due to its role in tissue tropism (Pallister et al., 1996). Hexon gene contain high amino acid variability between serotype and genotype for FAdV classification (Hess 2000;Meulemans et al., 2001). Hexon protein is made up of two pedestrial regions P1 and P2 and 4 loop regions L1 -L4 (Sheppard et al., 1995). L1 loop region being one of the projectiles is exposed, most variable and more prone to deletions and insertions than all the loop regions; and houses major deletions that leads to least homology of the virus ( Barua and Rai, 2004).
Epidemiological studies by few researchers revealed that the disease associated with FAdV especially inclusion body hepatitis (IBH), hydropericardium syndrome (HPS) and gizzard erosion (GE) have been increasing recently with major economic losses due to high mortality and poor productivity in infected farms (Cizmecigil et al., 2020;Cui et al., 2020). IBH is commonly caused by serotype 2, 8a, 8b and 11 (Kajan et al., 2013;Marek et al., 2016 while HPS and gizzard erosion are mainly caused by serotypes 4 and 1 respectively (Domanska-Blicharz et al., 2011;Grafl et al., 2015;Li et al., 2018). In Malaysia, IBH was first reported in state of Perak in 2005 caused by FAdV serotype 8b with a mortality of about 10% in broiler chickens (Hair-Bejo, 2005), and later with a mortality of 30% in sate of Johore and Malacca (Norina et al., 2016), the major states of poultry industry in the country. It is noteworthy that despite measures put in place by regulatory agencies against IBH in Malaysia which includes good management practices and vaccination against infectious bursal disease (IBD) and chicken anaemia virus (CAV) to control immunosuppression in poultry farms (Hair-Bejo, 2005;Juliana et al., 2014;Norina et al., 2016), IBH still poses a huge problem to the poultry industry. Vaccination then remains the control measure of choice in the management of IBH infections in Malaysia and worldwide (Schachner et al., 2021), but unavailability of commercialized suitable vaccine against FAdV infection is a hinderance. Furthermore, characterization of current FAdV isolates from IBH outbreak remain scanty in Malaysia which is crucial for effective control strategies. It was objective of the study to molecularly characterize FAdV isolates obtained from the field outbreaks of the disease in broiler chicken farms from 2017 to 2019.

Origin of the Isolates and Processing of Samples
Outbreaks of IBH were suspected in commercial broiler chickens aged 22 to 34-dayold from one farm in Sabah in 2017, and 3 farms in Johor in 2018 and 2019. chickens from infected farms were necrotized and gross lesion of liver and gizzard were noted. The liver and gizzard samples were collected and homogenized according to previous published protocol (Alemnesh et al., 2014) for molecular detection and characterisation of the virus. Briefly, the tissues were macerated with a sterile mortar and pestle in a 1 in 2 (w/v) suspensions in phosphate buffered saline (PBS, pH 7.4, 0.1M) prior centrifugation at 381 x g for 30 minutes. The supernatant was filtered through 0.45µm syringe filter and stored in -20˚C until use.
The FAdV isolates were identified as UPM1701, UPM1801, UPM1802, UPM1901 and UPM1902. The UPM1701 virus was isolated from liver of 34-day-old broiler chickens in Sabah in 2017. The UPM1801 and UPM1802 isolates were obtained from 22-day-old broiler chickens in a farm in Johor from two different houses in 2018. The affected chickens showed clinical signs of depression, weakness and lateral recumbence. Mortality of 2% was recorded within 2 to 3 days of the infection. The UPM1901 isolate was also obtained from Johore in 2019 from 25-day-old broiler chickens with history of 1.22% mortality. The UPM1902 isolate was also obtained from Johore from a 26day-old commercial broiler chickens with swollen and necrotized liver.

DNA Extraction, Conventional PCR and Agarose Gel Electrophoresis
The DNA from each sample was extracted using innuPREP Virus DNA kit (analytik jena, Germany) following manufacturer's recommended protocol. Measurement of DNA concentration was performed at wavelength 260/280nm by biophotometer (Eppendorf). Amplification of DNA nucleic acid was conducted by PCR using published universal hexon gene primer, HexA1/HexB1 (Meulemans et al., 2001;Marek et al., 2010;Norfitriah et al., 2018) in a total of 50µl PCR reaction using PCR Mastermix (Bioline, UK). The PCR products were separated in a 1% agarose gel electrophoresis using RedSafe™ Nuclei Acid Staining solution (iNtRON, Korea) and 1kb DNA Marker (GeneDirex, USA). Electrophoresis was conducted at 70 volts for 45 minutes prior visualization of DNA fragment band under U.V. transillumination.

DNA Purification, Sequencing and Nucleotide Sequence Analysis
All positive PCR product were purified by fragment DNA purification kit (iNtRON, Korea) according to manufacture procedure. The purified PCR products were submitted for sequencing (1st BASE, Singapore). Nucleotide sequences were retrieved, assembled and analyzed using Bioedit Version 7.2.5 software (Sohaimi et al., 2019). Online web based namely NCBI BLAST GenBank was used for sequence verification. Nucleotide sequence comparison between FAdV reference strains, UPM1137E2 and UPM04217 and the recent FAdV isolates were performed by multiple sequence alignment (MSA) using ClustalW program. The percentage of identity between isolates was carried out by sequence identity matrix in BioEdit package.

Phylogenetic Tree Analysis
Twenty-one published FAdV hexon gene sequences were obtained from GenBank database with accession numbers as follows: , KU517714 (UPM04217), JF917238 (UPM08158) and KF866370 (UPM1137E2). Phylogenetic tree was generated in MEGA version 5 software using Neighbour-joining method after MSA between recent FAdV isolates and 21 reference strains (Juliana et al., 2014).

Gross Lesions
The liver of UPM1701 was swollen, congested with mild multifocal areas of necrosis and petechial haemorrhages. The UPM1801 and UPM1802 livers were also swollen and yellowish with mild area of petechial haemorrhages. The liver of UPM1901 was pale and swollen with areas of necrosis and haemorrhages. Erosion at the koilin layer of gizzard and enlargement of the proventriculus were also recorded ( Figure 1). The UPM1902 showed swollen and necrotized liver.

Nucleotide and Amino Acid Sequences Analysis
Nucleotide (nt) sequences of partial hexon gene with length 596 bases for recent isolates were confirmed as Fowl adenovirus in NCBI Blast which corresponds to the L1 loop region of hexon. The percentage identity value is highest for UPM1801 and UPM1802 isolates with 100% identity (E value = 0.0) to reference strain, UPM04217 from serotype 8b species E. There are 98% to 99% identity between UPM1701, UPM1901 and UPM1902 to reference strains under serotype 8b in GenBank databases.
Multiple sequence alignment of 596bp nucleotide sequences in L1 loop region region on hexon gene between recent FAdV isolates and Malaysian`s FAdV reference strains, UPM1137E2 and UPM04217 (Figure 3). These nucleotides translated to 198 amino acids ( Figure  4). There were 31 nucleotide base substitutions among the sequences of isolates UPM1701, UPM1901 and UPM1902 compared to the earlier reference Malaysian isolates in GenBank. Out of these changes 18 were synonymous while 13 were non-synonymous which yielded 10 amino acid substitutions. These includes I► 40 V, D► 43 E, G► 44 D, T► 48 S, A► 61 Q, A► 70 G, G► 87 T, L► 127 I, G► 178 R and E► 179 A. These changes were not observed among the UPM1801 and UPM1802 isolates and could singly or in combination play a role in pathogenicity of the isolates.
Sequence identity matrix revealed 100% identities among UPM1701, UPM1901 and UPM1902 isolates on entire nucleotide sequence length (Table 1). Comparison those three isolates with UPM1801 and UPM1802 isolates showed 94.8% identity. The percentage identity between current isolates and Malaysian reference strains, UPM1137E2 and UPM04217 revealed highest identity to both UPM1801 and UPM1802 with 99.3% and 99.6% identity, respectively. For UPM1701, UPM1901 and UPM1902 isolates, the identity was 94.7% and 95.1% identity compared to UPM1137E2 and UPM04217, respectively.

Phylogenetic Tree Analysis
Phylogenetic tree of 26 aligned nucleotide bases on partial hexon gene revealed FAdV classification into 5 species designated as letter A to E as differentiated by colour indicator (Figure 5). Recent FAdV isolates, UPM1701, UPM1801, UPM1802, UPM1901 and UPM1902 were classified under species E serotype 8b and shared common ancestor with UPM04217, UPM1137E2 and 764 strains.

DISCUSSION
The FAdV serotype 8b has been reported to be the most prominent cause of IBH in broiler chickens worldwide leading to significant economic losses in poultry industry due to high mortality, low productivity and performance (Grafl et al., 2012;Lim et al., 2012;Kajan et al., 2013;Morshed et al., 2017). The gross lesions recorded in the present IBH cases which include swollen and yellowish liver with areas of haemorrhages and necrosis are consistent with IBH cases reported previously (Kumar et al., 2013;Maartens et al., 2014). But the presence of gizzard erosion in of this case (UPM1901) which was more commonly recorded in serotype 1 of the virus, but not for the serotype 8b is noteworthy and could indicate increase in pathogenic effects. Severe gizzard erosion was previously reported in a case of IBH outbreaks (UPM1137) involving 25 to 27 weeks old layer chickens in the country in 2011 in which intranuclear inclusion bodies and FAdV serotype 8b were detected both in the liver and gizzard (Norfitriah et al., 2018). The pathogenicity of FAdVs are highly related to virulence determinant gene which is located in L1 loop of hexon gene (Marek et al., 2010;Sohaimi et al., 2019). Liver is the major predilection site for FAdV replication where it causes destruction of tissue based on necropsy findings, but gizzard could also be affected (Steer et al., 2015).
All the 5 FAdV isolates in the present study were closely related to UPM1137E2 reference strain obtained from previous concurrent IBH infection with gizzard erosion (Sohaimi et al., 2018). The hexon gene primers used in this study were previously used successfully to detect 12 serotypes of FAdV as reported in previous finding (Meulemans et al., 2001) and yielded   (Raue et al., 1998;Ojkic et al., 2008). It indicates that FAdV serotype 8b could be the sole causative agent of IBH outbreaks in Malaysia and is highly pathogenic in chickens as previously reported (Hair-Bejo, 2005;Norina et al., 2016;Norfitriah et al., 2019).
The phylogenetic tree of 5 hexon nucleotide sequences obtained from the present 5 IBH cases and 21 reference strains in GenBank derived from 12 serotypes confirmed that the recent isolates belong to FAdV serotype 8b share common ancestory with reference strains from Malaysia (UPM1137E2 and UPM04217) and high pathogenic strain 764 from United Kingdom (Dar et al., 2012). This has shown that it is probably FadV 8b from same origin that has been circulating in malaysia, and it seems the isolate is evolving into higher pathogenicity across outbreaks. This was shown in the occurrence of gizzard erosion in this IBH case associated with FadV 8b. This calls for stringent preventive measures in Malaysia and IBH endemic regions because the spread of FAdV 8b could follow the Indian experience where the first outbreak of FAdV occured in 1994 in some parts of Jammu and Kashmir, Punjab and Delhi (Gowda and Satyanarayana, 1994), later spread to Uttar Pradesh same year (Kumar et al., 1997) and then nationwide (Bhowmik, 1996;Nighot et al., 1996;Asrani et al., 1997).
There was a total of 31 nucleotide substitutions which translated to 10 amino acid mutations among isolates UPM1701 (Sabah), UPM1901 and UPM1902 (Johore) when compared to UPM04217 and UPM08158 isolated in 2004 and 2008 respectively (Juliana et al., 2014), and UPM1137E2 isolated in 2011 (Sohaimi et al., 2018). But these changes were not observed in UPM1801 and UPM1802 isolates which have closer similarity to the earlier isolates. This has demonstrated the occurrence of the FAdV serotype 8b divergence or mutation since the first outbreak of the disease reported in 2005 (Hair-Bejo, 2005). Divergence in FadV 8b genome occuring in different outbreaks has not been reported in Malaysia before and this is coming with novel lesion (gizard erosion) that is not associated with serotype 8b infections which is worrisome. Viruses are always involved with changes in    references strains retrieved from GenBank database. The recent isolates, UPM1701, UPM1801, UPM1802, UPM1901 and UPM1902 were classified under species E and belong to serotype 8b FAdV. Color indicator : 1) Blue = Group E, 2) Red = Group D, 3) Yellow = Group C, 4) Black = Group B, 5) Green = Group A their genome due to genetic selection that could lead to mutations; and mutations could be detrimental, beneficial or of no effect (Fleischmann, 1996). It could produce viruses with increased or reduced pathogenicity, changed host range or tissue tropism (Fleischmann, 1996). Since FAdV infection are spreading to various areas, development of vaccines is crucial and understanding the genetic diversities is very important in designing appropriate vaccines for prevention and control of IBH. Although adequate biosecurity measures could be of help in limiting the spread of IBH since horizontal transfer is an established transmission mechanism (McFerran and Adair, 2003), vaccination using inactivated or attenuated FadV as established elsewhere (Steer et al., 2009;Kim et al., 2014;Schachner et al., 2018), could be the best method of control and eradication. However, following the discovery of molecular changes in recent isolates, regular monitoring of successive outbreaks is highly recommended in endemic areas of the world to safeguard against vaccine failure where vaccines exist and guide in development of appropriate vaccines where it is currently lacking.

CONCLUSION
The recent isolates of FadV were serotype 8b and were the cause of the IBH outbreaks in the poultry farms in Malaysia. These outbreaks occurred with gizzard erosion in one case which is uncommon with FadV serotype 8b. There were also nucleotide changes and amino acid mutations in the isolates from different regions of the country which could be important in the pathogenicity of the virus and could be helpful in the design of vaccine for prevention and control of IBH disease.