Molecular Characterization of Peruvian Fowl Adenovirus (FAdV) Isolates

Forty seven clinical samples of Fowl adenovirus (FAdV) associated with Inclusion Body Hepatitis (IBH) from Peruvian broilers received between July 2006 and April 2013 were genotyped using sequencing of L1 Loop of Hexon gene. All 47 clinical samples presented macroscopic and histopathology lesions consistent with IBH, and amplified a specific fragment of Hexon gene by Polymerase Chain Reaction (PCR). A unique nucleotide sequence of 789 base pairs of Hexon gene (position 273 to 1061) was obtained in all 47 clinical samples analyzed. This sequence showed a high level of conservation in amino acid and nucleotide sequence (>99%) with a Fowl Adenovirus C serotype 4 previously identified. Sequence and phylogenetic analysis indicate no genotypic variation in Peruvian isolates. The presence of a unique genotype very closely related with genotype C1 previously reported in Peru and Ecuador (>99%), suggests the presence of FAdV C serotype 4 genotype C1 in clinical cases of IBH from Peruvian broilers.


Introduction
Fowl Adenovirus (FAdV) includes 12 serotypes [1], with a high diversity of strains for each serotype and a wide range of clinical and pathological presentations (pneumonias, tracheitis, gizzard erosions, proventriculitis, pancreatitis, inclusion body hepatitis and hydropericardium) [2].The most important sanitary problem in North America and South America is inclusion body hepatitis/hydropericardium syndrome due to high levels of morbidity and mortality in broilers resulting in significant economic losses [3]- [7].This great diversity exhibited by FAdV required the use of additional techniques to make a correct identification of levels of species and serotypes and characterization of genetic diversity.An alternative to microbiologic and serologic techniques are molecular techniques based on DNA analysis.Several studies have reported the feasibility of molecular genetic tools for genetic differentiation and molecular typing of FAdV.The use of PCR for Hexon gene, RFLP analysis and direct sequencing of Hexon and Penton genes [8]- [10] have permitted a correct identification and characterization of clinical isolates.The lack of information available for molecular characterization of isolates from Peru do not allow the identification of the type of serotypes currently present in poultry production systems.This is a key requirement for establishing actions to control and prevent IBH.The objective of this study was to determine FAdV genotypes/serotypes isolated from IBH clinical cases in broilers from Peru.

Sources of Samples
A total of 47 suspected clinical cases of Inclusion body hepatitis (IBH) from broilers received in the Microbiology Laboratory-Bioservice SRL between July 2006 and April 2013 were included in this study (Table 1).Agar gel immune-diffusion (AGID) tests for serotypes 4, 8 and 9, liver histopathology analysis and Hexon gene PCR were performed to confirm the presence of fowl adenovirus (FAdV) in all suspected clinical cases.

Viral DNA Extraction and PCR Amplification of Hexon Gene
Viral DNA was extracted from liver lysate using DNeasy Tissue kit (Qiagen) according to manufacturer.PCR for L1 loop of the Hexon gene was carried out using primers HA and HB as described previously [9].In brief, the PCR reaction was performed using a final volume of 20 uL containing 10 ng genomic DNA, 1X PCR buffer (20 mM Tris-HCl, 20 mM KCl, 5 mM (NH4) 2 SO 4 ), 2 mM MgCl2, 5 pmol of each primer, 0.2 mM of dNTPs, 0.5 U Maxima HotStart-Taq DNA polymerase (Fermentas).Thermal cycles were the following: initial denaturation of 94˚C for 3 minutes, 30 cycles of 94˚C for 30 seconds, 53˚C for 60 seconds, 72˚C for 30 seconds and a final extension of 72˚C for 5 minutes.The PCR amplification was carried out using a Veriti Thermal cycler (Applied Biosystems).The PCR products were separated in 1% agarose gel TBE 1X electrophoresis and visualized by fluorescence using an ethidium bromide solution (0.1 ug/mL) and UV light.

Sequencing and Phylogenetics Analysis of L1 Loop of Hexon Gene
The PCR products were purified using the Wizard ® SV Gel and PCR Clean-Up kit, cloned with pGEM-T Easy (Promega) and submitted for sequencing to Macrogen Inc, Korea.The sequencing reaction was performed in both directions using Big Dye sequencing kit (Applied Biosystems) and the primers HA and HB.Partial nucleotide sequence of the L1 Loop of hexon gene from FAdV's was submitted to Gene Bank with the following accession number: KF601685.
Forward and reverse sequences were aligned together using Clustal program [11] to create consensus sequences.The consensus nucleotide sequences were aligned by the Clustal W method using MEGA 5.0 software [12].Pairwise amino acid and nucleotide identity and pairwise evolutionary distances were computed using using Kimura-2-parameter method [13] and Dayhoff matrix based method [14].A dendogram was constructed with the Neighbor-joining method [15] using 1000 bootstrap [16] replicate values with Mega v5.0.

Clinical Cases of Inclusion Body Hepatitis
All clinical cases presented liver histopathology consistent with IBH lesions hepatocyte cytoplasmic vacuolation, multifocal to coalescing areas of hepatocytes necrosis, irregular shape hepatocyte nuclei, presence of basophilic intranuclear inclusion bodies without presence of HPS.The presence of FAdV was confirmed by PCR amplification of Hexon gene fragment in 47 suspected clinical cases of IBH.The evolutionary relationships of taxa were inferred using Neighbor-Joining method [17].The evolutionary distances were computed using the Kimura 2-parameter method [13].The analysis involved 20 nucleotide sequences and 770 nucleotide positions in the final dataset, the strain names are according to Table 2. Bootstrap [16] test (1000 replicates) is expressed in % values.The scale bar indicates the evolutionary distance between sequences; (b) Phylogenetic tree of L1 loop of the hexon protein sequence from 12 species of FAdV's.The evolutionary relationships of taxa were inferred using Neighbor-Joining method [17].The evolutionary distances were computed using the Dayhoff matrix method [14].The analysis involved 20 amino acid sequences and 199 amino acid positions in the final dataset, the strain names are according to

Discussion
The nucleic acid technology has demonstrated the utility to detect differences in structural proteins related with immune response as Hexon protein.The sequencing of Hexon gene permits a correct identification of species level and serotype in FAdV, with similar results as reported for RFLP genome analysis [2].Inside Hexon gene, 7 hypervariable regions were identified exclusively located in Loop 1 (L1) and Loop 2 (L2) regions [20].The loop 1 in Hexon gene of FAdV reported higher variability [8], and has demonstrated additional applications than include genetic variability analysis of FAdV isolates [6] [10] [21].The evolutionary relationships of taxa were inferred using Neighbor-Joining method [17].The evolutionary distances were computed using the Kimura 2-parameter method [13].The analysis involved 19 nucleotide sequences and 611 nucleotide positions in the final dataset, the strain names are according to Table 2. Bootstrap [16] test (1000 replicates) is expressed in % values.The scale bar indicates the evolutionary distance between sequences.(b) Phylogenetic tree of L1 loop of the hexon protein sequence from FAdV C serotype 4. The evolutionary relationships of taxa were inferred using Neighbor-Joining method [17].The evolutionary distances were computed using the Dayhoff matrix method [14].The analysis involved 19 amino acid sequences and 202 amino acid positions in the final dataset, the strain names are according to Table 2. Bootstrap [16] test (1000 replicates) is expressed in % values.The scale bar indicates the evolutionary distance between sequences.
During this study we analyzed 47 FAdV isolates from IBH clinical cases, examined their Hexon gene loop 1 sequences and compared them with reference strains previously published sequences.Our results suggest the absence of genetic variability in FAdV C in Peru, similar to previous reports [19]; however this study only has access to one Peruvian isolate.Similar results were reported from India [22] [23] and South Korea [24] for clinical cases of IBH associated with the presence of FAdV C serotype 4 (>99% clinical cases of IBH).However, FAdV C serotype 4 (strain K531) is also associated with hydropericardium syndrome outbreaks (HPS) from South Korea [24] [25] and Germany [26] in contrast with Peruvian FAdV C serotype 4 than only reports IBH clinical lesions.The amino acid sequence analysis shows a slight differentiation between isolates from Peru and South Korea (Table 4) and phylogenetic analysis corroborates the different genetic clustering (Figure 2(B)).

Conclusion
In conclusion, our results indicate the presence of FAdV C, serotype 4, genotype C1 circulating in clinical cases of IBH from Peruvian broilers.

Figure 1 .
Figure 1.(a) Phylogenetic tree of L1 loop of the hexon nucleotide sequence from 12 species of FAdV's.The evolutionary relationships of taxa were inferred using Neighbor-Joining method[17].The evolutionary distances were computed using the Kimura 2-parameter method[13].The analysis involved 20 nucleotide sequences and 770 nucleotide positions in the final dataset, the strain names are according to Table2.Bootstrap[16] test (1000 replicates) is expressed in % values.The scale bar indicates the evolutionary distance between sequences; (b) Phylogenetic tree of L1 loop of the hexon protein sequence from 12 species of FAdV's.The evolutionary relationships of taxa were inferred using Neighbor-Joining method[17].The evolutionary distances were computed using the Dayhoff matrix method[14].The analysis involved 20 amino acid sequences and 199 amino acid positions in the final dataset, the strain names are according to Table2.Bootstrap[16] test (1000 replicates) is expressed in % values.The scale bar indicates the evolutionary distance between sequences.

Figure 2 .
Figure 2. (a) Phylogenetic tree of L1 loop of the hexon nucleotide sequence from FAdV C serotype 4.The evolutionary relationships of taxa were inferred using Neighbor-Joining method[17].The evolutionary distances were computed using the Kimura 2-parameter method[13].The analysis involved 19 nucleotide sequences and 611 nucleotide positions in the final dataset, the strain names are according to Table2.Bootstrap[16] test (1000 replicates) is expressed in % values.The scale bar indicates the evolutionary distance between sequences.(b) Phylogenetic tree of L1 loop of the hexon protein sequence from FAdV C serotype 4. The evolutionary relationships of taxa were inferred using Neighbor-Joining method[17].The evolutionary distances were computed using the Dayhoff matrix method[14].The analysis involved 19 amino acid sequences and 202 amino acid positions in the final dataset, the strain names are according to Table2.Bootstrap[16] test (1000 replicates) is expressed in % values.The scale bar indicates the evolutionary distance between sequences.

Table 1 .
Collection year and city of origin of 47 clinical cases of Inclusion body hepatitis (IBH) from broilers from Peru.

of isolation Place of isolation (City, Province) Number of isolates
Mega v5.0.A phylogenetic analysis for the distance method was performed using partial sequences of L1 loop of Hexon gene and Hexon protein previously published in Gene Bank and FAdV Peruvian sequence (Table2)

Table 2 .
ID sequence, accession number, country of origin, species, serotype and genetic cluster of 37 FAdV using phylogenetic analysis.

Table 3
[10]47 Peruvian FAdV isolates did not show genotype variation, a unique nucleotide sequence of 789 bp corresponding to position 273 and 1061 of Hexon gene amplified showed high nucleotide identity level (99.5% identity, E value = 0) and high conservation level in the amino acidic sequence (99.5%) in relation with a FAdV C serotype 4 previously identified[10].The pairwise matrix of evolutionary divergence (nucleotide and amino acid) showed less divergence between Peruvian FAdV with FAdV C serotype 4 (0.005, 0.005) and FAdV C serotype US 9 (0.029, 0.070) in contrast to FAdV D serotype 8 (0.365, 0.709) and serotype 12 (0.378 and 0.663) ( ). Phylogenetic analysis from a segment of 770 nucleotide (260 amino acids) suggests the presence of FAdV group C serotype 4 present in all clinical cases from IBH of Peruvian broilers (Figure1(a) and

Table 2 .
Bootstrap [16]test (1000 replicates) is expressed in % values.The scale bar indicates the evolutionary distance between sequences.

Table 3 .
Pairwise estimates of evolutionary divergence 1 of nucleotide sequence (below diagonal) and amino acid sequence (above diagonal) between 12 species of FAdV 2 .

Table 4 .
Pairwise estimates of evolutionary divergence 1 of nucleotide sequences (below diagonal) and amino acid sequence (above diagonal) between 19 strain of FAdV-C serotype 4.