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.
Fowl Adenovirus (FAdV) includes 12 serotypes [
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 (
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 [
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 [
. Collection year and city of origin of 47 clinical cases of Inclusion body hepatitis (IBH) from broilers from Peru
Year of isolation | Place of isolation (City, Province) | Number of isolates |
---|---|---|
2006, 2007, 2011, 2012, 2013 | Lima, Lima | 12 |
2011, 2012, 2013 | Huaral, Lima | 20 |
2011, 2012, 2013 | Chilca, Lima | 1 |
2011 | Iquitos, Loreto | 2 |
2011 | Arequipa, Arequipa | 1 |
2011 | Tacna, Tacna | 1 |
2011 | Chanchamayo, Junin | 2 |
2011 | Nazca, Ica | 1 |
2006, 2007, 2012, 2013 | Trujillo, La Libertad | 7 |
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 (
. ID sequence, accession number, country of origin, species, serotype and genetic cluster of 37 FAdV using phylogenetic analysis
ID Sequence | Accession number1 | Country2 | Species3 | Serotype4 | Genetic Cluster5 |
---|---|---|---|---|---|
FAdV_1_(CELO) | AAL13217 | NR6 | A | EU/US 1 | A |
FAdV_2_(SR48) | AAN77072 | NR6 | D | EU 2 | D2 |
FAdV_3_(75) | AAN77075 | NR6 | D | EU 3 | D3 |
FAdV_4_(KR5) | AAN77077 | NR6 | C | EU 4 | C |
FAdV_5_(VR-830) | AAL13222 | NR6 | E | US 5 | D1 |
FAdV_5_(TR22) | AAN77079 | NR6 | B | EU 5 | E |
FAdV_5_(340) | AAN77078 | NR6 | B | EU 5 | B |
FAdV_6_(VR-831) | AAL13224 | NR6 | D | US 6 | D3 |
FAdV_6_(CR119) | AAN77080 | NR6 | E | EU 6 | D1 |
FAdV_7_(YR36) | AAN77081 | NR6 | E | EU 7 | D1 |
FAdV_7_(VR-832) | AAL13225 | NR6 | E | EU 7 | D1 |
FAdV_8_(TR59) | AAN77082 | NR6 | E | EU 8 | D1 |
FAdV_8_(VR-833) | AAL13221 | NR6 | D | US 8 | D3 |
FAdV_9_(VR-834) | AAL13226 | NR6 | C | US 9 | C |
FAdV_9_(764) | AAN77084 | NR6 | E | EU 9 | D1 |
FAdV_10_(VR-835) | AAL13227 | NR6 | E | US 10 | D1 |
FAdV_11_(X11) | AAL13223 | NR6 | E | US 11 | D1 |
FAdV_11_(C2B) | AAN77085 | NR6 | C | EU 11 | D2 |
FAdV_12_(380) | AAL13228 | NR6 | D | US 12 | D2 |
(922-1)_Germany | FN869978.1 | Germany | C | EU/US 4 | C |
(09-2602)_Austria | FN869977.1 | Austria | C | EU/US 4 | C |
(K31)_Pakistan | FN869976.1 | Pakistan | C | EU/US 4 | C |
(09-584)_Austria | FN869975.1 | Austria | C | EU/US 4 | C |
(53)_Peru | FN869973.1 | Peru | C | EU/US 4 | C |
(K1013)_Ecuador | FN869972.1 | Ecuador | C | EU/US 4 | C |
(Da60)_Germany | FN869971.1 | Germany | C | EU/US 4 | C |
(K99-97)_Kuwait | FN869970.1 | Kuwait | C | EU/US 4 | C |
(AG234)_Mexico | FN869969.1 | Mexico | C | EU/US 4 | C |
South_Korea | HQ697593.1 | South Korea | C | EU/US 4 | C |
(04-50388)_Canada | EF685395.1 | Canada | C | EU/US 4 | C |
(488)_Russia | AY581295.1 | Russia | C | EU/US 4 | C |
(4158)_Italy | HM592284.1 | Italy | C | EU/US 4 | C |
(5997)_Italy | HM592281.1 | Italy | C | EU/US 4 | C |
(6169)_Italy | HM592277.1 | Italy | C | EU/US 4 | C |
(5670)_Italy | HM592274.1 | Italy | C | EU/US 4 | C |
(488)_Indian | AY581295.1 | India | C | EU/US 4 | C |
(HARYANA-07)_Indian | EU847626.1 | India | C | EU/US 4 | C |
1Accesion number for Hexon protein or sequence in database Genbank or EMBL. 2Country of origin of strain. 3Fowl Adenovirus species according with [
using Kimura-2-parameter method [
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.
All 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 [
(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 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 Table 2. Bootstrap [16] test (1000 replicates) is expressed in % values. The scale bar indicates the evolutionary distance between sequences
. Pairwise estimates of evolutionary divergence1 of nucleotide sequence (below diagonal) and amino acid sequence (above diagonal) between 12 species of FAdV2
FAdV Peru | FAdV1 | FAdV2 | FAdV3 | FAdV4 | FAdV5A | FAdV5B | FAdV5C | FAdV6A | FAdV6B | FAdV7A | FAdV7B | FAdV8A | FAdV8B | FAdV9A | FAdV9B | FAdV10 | FAdV11A | FAdV11B | FAdV12 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
FAdVPeru | 0.628 | 0.663 | 0.704 | 0.005 | 0.603 | 0.663 | 0.573 | 0.704 | 0.568 | 0.578 | 0.573 | 0.548 | 0.709 | 0.070 | 0.603 | 0.598 | 0.583 | 0.663 | 0.663 | |
FAdV1 | 0.300 | 0.573 | 0.608 | 0.628 | 0.583 | 0.648 | 0.598 | 0.608 | 0.603 | 0.553 | 0.563 | 0.578 | 0.603 | 0.618 | 0.583 | 0.598 | 0.553 | 0.563 | 0.563 | |
FAdV2 | 0.374 | 0.335 | 0.432 | 0.663 | 0.558 | 0.663 | 0.563 | 0.432 | 0.508 | 0.543 | 0.528 | 0.528 | 0.427 | 0.658 | 0.558 | 0.553 | 0.538 | 0.030 | 0.030 | |
FAdV3 | 0.366 | 0.323 | 0.203 | 0.704 | 0.568 | 0.588 | 0.588 | 0.000 | 0.588 | 0.563 | 0.558 | 0.578 | 0.101 | 0.724 | 0.568 | 0.573 | 0.563 | 0.417 | 0.417 | |
FAdV4 | 0.005 | 0.299 | 0.374 | 0.365 | 0.603 | 0.663 | 0.573 | 0.704 | 0.568 | 0.578 | 0.573 | 0.548 | 0.709 | 0.070 | 0.603 | 0.598 | 0.583 | 0.663 | 0.663 | |
FAdV5A | 0.353 | 0.321 | 0.278 | 0.273 | 0.352 | 0.573 | 0.543 | 0.568 | 0.317 | 0.216 | 0.246 | 0.271 | 0.563 | 0.613 | 0.010 | 0.035 | 0.221 | 0.563 | 0.563 | |
FAdV5B | 0.361 | 0.340 | 0.303 | 0.264 | 0.360 | 0.278 | 0.533 | 0.588 | 0.573 | 0.558 | 0.568 | 0.543 | 0.598 | 0.653 | 0.573 | 0.568 | 0.558 | 0.653 | 0.653 | |
FAdV5C | 0.332 | 0.312 | 0.295 | 0.305 | 0.331 | 0.284 | 0.271 | 0.588 | 0.503 | 0.523 | 0.518 | 0.528 | 0.583 | 0.588 | 0.543 | 0.538 | 0.518 | 0.563 | 0.563 | |
FAdV6A | 0.369 | 0.326 | 0.205 | 0.003 | 0.368 | 0.275 | 0.266 | 0.308 | 0.588 | 0.563 | 0.558 | 0.578 | 0.101 | 0.724 | 0.568 | 0.573 | 0.563 | 0.417 | 0.417 | |
FAdV6B | 0.339 | 0.313 | 0.240 | 0.258 | 0.339 | 0.151 | 0.279 | 0.262 | 0.261 | 0.286 | 0.281 | 0.302 | 0.573 | 0.563 | 0.322 | 0.312 | 0.286 | 0.533 | 0.533 | |
FAdV7A | 0.336 | 0.301 | 0.262 | 0.268 | 0.336 | 0.096 | 0.278 | 0.265 | 0.270 | 0.117 | 0.055 | 0.231 | 0.568 | 0.578 | 0.221 | 0.211 | 0.010 | 0.553 | 0.553 | |
FAdV7B | 0.331 | 0.305 | 0.257 | 0.264 | 0.332 | 0.105 | 0.277 | 0.264 | 0.266 | 0.116 | 0.021 | 0.266 | 0.568 | 0.573 | 0.241 | 0.241 | 0.065 | 0.543 | 0.543 | |
FAdV8A | 0.339 | 0.310 | 0.249 | 0.274 | 0.339 | 0.145 | 0.270 | 0.268 | 0.277 | 0.140 | 0.122 | 0.135 | 0.573 | 0.543 | 0.271 | 0.256 | 0.236 | 0.533 | 0.533 | |
FAdV8B | 0.365 | 0.323 | 0.200 | 0.044 | 0.364 | 0.271 | 0.275 | 0.308 | 0.047 | 0.260 | 0.265 | 0.264 | 0.279 | 0.714 | 0.563 | 0.573 | 0.568 | 0.412 | 0.412 | |
FAdV9A | 0.029 | 0.301 | 0.371 | 0.368 | 0.031 | 0.353 | 0.355 | 0.334 | 0.370 | 0.340 | 0.339 | 0.336 | 0.338 | 0.364 | 0.613 | 0.613 | 0.583 | 0.668 | 0.668 | |
FAdV9B | 0.355 | 0.322 | 0.277 | 0.271 | 0.353 | 0.009 | 0.278 | 0.283 | 0.274 | 0.151 | 0.094 | 0.100 | 0.145 | 0.271 | 0.355 | 0.035 | 0.226 | 0.563 | 0.563 | |
FAdV10 | 0.356 | 0.327 | 0.274 | 0.274 | 0.355 | 0.021 | 0.278 | 0.283 | 0.277 | 0.144 | 0.090 | 0.099 | 0.138 | 0.273 | 0.357 | 0.021 | 0.216 | 0.568 | 0.568 | |
FAdV11A | 0.338 | 0.300 | 0.260 | 0.266 | 0.338 | 0.097 | 0.277 | 0.262 | 0.269 | 0.117 | 0.003 | 0.023 | 0.123 | 0.264 | 0.340 | 0.095 | 0.091 | 0.548 | 0.548 | |
FAdV11B | 0.378 | 0.332 | 0.010 | 0.199 | 0.378 | 0.281 | 0.296 | 0.295 | 0.201 | 0.248 | 0.269 | 0.262 | 0.252 | 0.196 | 0.378 | 0.279 | 0.277 | 0.266 | 0.000 | |
FAdV12 | 0.378 | 0.335 | 0.010 | 0.201 | 0.378 | 0.282 | 0.299 | 0.296 | 0.204 | 0.248 | 0.270 | 0.262 | 0.253 | 0.199 | 0.378 | 0.281 | 0.278 | 0.268 | 0.003 |
1Analyses were conducted using p-distance [
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 [
(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 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 [
The presence of multiple FAdV species in clinical cases of IBH were reported in Canada [
This study is the first report of genetic characterization of FAdV C isolates from Peru that includes samples from a coastal region (Ica, La Libertad and Lima regions) which concentrates 80% of poultry production systems in Peru and provides important information than can serve as starting point for further investigations related to pathogenicity, antigenic properties and specific vaccine development for prevention and control of IBH
. Pairwise estimates of evolutionary divergence1 of nucleotide sequences (below diagonal) and amino acid sequence (above diagonal) between 19 strain of FAdV-C serotype 4
FAdV Peru | Germany A | Austria A | Pakistan | Austria B | Peru | Ecuador | Germany B | Kuwait | Mexico | South Korea | Canada | Russia A | Italy A | Italy B | Italy C | Italy D | Indian A | Indian B | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
FAdV Peru | 0.050 | 0.045 | 0.035 | 0.045 | 0.000 | 0.000 | 0.010 | 0.005 | 0.005 | 0.025 | 0.010 | 0.035 | 0.030 | 0.045 | 0.010 | 0.025 | 0.035 | 0.040 | |
Germany A | 0.029 | 0.005 | 0.015 | 0.010 | 0.050 | 0.050 | 0.040 | 0.045 | 0.045 | 0.045 | 0.040 | 0.015 | 0.030 | 0.005 | 0.045 | 0.030 | 0.015 | 0.020 | |
Austria A | 0.023 | 0.008 | 0.010 | 0.005 | 0.045 | 0.045 | 0.035 | 0.040 | 0.040 | 0.040 | 0.035 | 0.010 | 0.025 | 0.000 | 0.040 | 0.025 | 0.010 | 0.015 | |
Pakistan | 0.023 | 0.008 | 0.007 | 0.010 | 0.035 | 0.035 | 0.035 | 0.030 | 0.030 | 0.050 | 0.035 | 0.000 | 0.020 | 0.010 | 0.035 | 0.020 | 0.000 | 0.005 | |
Austria B | 0.026 | 0.011 | 0.007 | 0.010 | 0.045 | 0.045 | 0.040 | 0.040 | 0.040 | 0.045 | 0.040 | 0.010 | 0.020 | 0.005 | 0.035 | 0.020 | 0.010 | 0.015 | |
Peru | 0.000 | 0.029 | 0.023 | 0.023 | 0.026 | 0.000 | 0.010 | 0.005 | 0.005 | 0.025 | 0.010 | 0.035 | 0.030 | 0.045 | 0.010 | 0.025 | 0.035 | 0.040 | |
Ecuador | 0.002 | 0.031 | 0.025 | 0.025 | 0.028 | 0.002 | 0.010 | 0.005 | 0.005 | 0.025 | 0.010 | 0.035 | 0.030 | 0.045 | 0.010 | 0.025 | 0.035 | 0.040 | |
Germany B | 0.007 | 0.028 | 0.020 | 0.023 | 0.023 | 0.007 | 0.008 | 0.005 | 0.005 | 0.015 | 0.000 | 0.035 | 0.025 | 0.035 | 0.005 | 0.020 | 0.035 | 0.040 | |
Kuwait | 0.005 | 0.029 | 0.021 | 0.021 | 0.025 | 0.005 | 0.007 | 0.002 | 0.000 | 0.020 | 0.005 | 0.030 | 0.025 | 0.040 | 0.005 | 0.020 | 0.030 | 0.035 | |
Mexico | 0.007 | 0.031 | 0.023 | 0.023 | 0.026 | 0.007 | 0.008 | 0.003 | 0.002 | 0.020 | 0.005 | 0.030 | 0.025 | 0.040 | 0.005 | 0.020 | 0.030 | 0.035 | |
South Korea | 0.016 | 0.028 | 0.026 | 0.026 | 0.026 | 0.016 | 0.018 | 0.010 | 0.011 | 0.013 | 0.015 | 0.050 | 0.040 | 0.040 | 0.020 | 0.035 | 0.050 | 0.054 | |
Canada | 0.008 | 0.029 | 0.021 | 0.025 | 0.025 | 0.008 | 0.010 | 0.002 | 0.003 | 0.005 | 0.011 | 0.035 | 0.025 | 0.035 | 0.005 | 0.020 | 0.035 | 0.040 | |
Russia A | 0.023 | 0.008 | 0.007 | 0.000 | 0.010 | 0.023 | 0.025 | 0.023 | 0.021 | 0.023 | 0.026 | 0.025 | 0.020 | 0.010 | 0.035 | 0.020 | 0.000 | 0.005 | |
Italy A | 0.025 | 0.020 | 0.018 | 0.015 | 0.015 | 0.025 | 0.026 | 0.021 | 0.023 | 0.025 | 0.021 | 0.023 | 0.015 | 0.025 | 0.020 | 0.010 | 0.020 | 0.025 | |
Italy B | 0.026 | 0.005 | 0.003 | 0.003 | 0.007 | 0.026 | 0.028 | 0.023 | 0.025 | 0.026 | 0.023 | 0.025 | 0.003 | 0.015 | 0.040 | 0.025 | 0.010 | 0.015 | |
Italy C | 0.011 | 0.026 | 0.021 | 0.021 | 0.025 | 0.011 | 0.013 | 0.005 | 0.007 | 0.008 | 0.011 | 0.007 | 0.021 | 0.016 | 0.021 | 0.015 | 0.035 | 0.040 | |
Italy D | 0.021 | 0.020 | 0.018 | 0.015 | 0.015 | 0.021 | 0.023 | 0.018 | 0.020 | 0.021 | 0.018 | 0.020 | 0.015 | 0.003 | 0.015 | 0.013 | 0.020 | 0.025 | |
Indian A | 0.023 | 0.008 | 0.007 | 0.000 | 0.010 | 0.023 | 0.025 | 0.023 | 0.021 | 0.023 | 0.026 | 0.025 | 0.000 | 0.015 | 0.003 | 0.021 | 0.015 | 0.005 | |
Indian B | 0.025 | 0.010 | 0.008 | 0.002 | 0.011 | 0.025 | 0.026 | 0.025 | 0.023 | 0.025 | 0.028 | 0.026 | 0.002 | 0.016 | 0.005 | 0.023 | 0.016 | 0.002 |
1Analyses were conducted using p-distance [
associated with FAdV.
In conclusion, our results indicate the presence of FAdV C, serotype 4, genotype C1 circulating in clinical cases of IBH from Peruvian broilers.
This work was supported by Bioservice SRL. The authors wish to thanks to Dr. Karina Mendoza, Dr. Katherine Porturas, Dr. Stephane Lovon, Dr. Brenda Jara, Dr. Madeline Garcia, Dr. Heberht Uchuya and Dr. Jhonatan Castro for their cooperation in the collection and isolation of viral samples. We would like to also thank Dr. Manuel Moro for editorial comments and suggestions.
*Corresponding author.