J Vet Sci.  2018 Nov;19(6):759-770. 10.4142/jvs.2018.19.6.759.

Hexon and fiber gene changes in an attenuated fowl adenovirus isolate from Malaysia in embryonated chicken eggs and its infectivity in chickens

• Sohaimi NM
• Bejo MH
• Omar AR
• Ideris A
• Isa NM

• KMID: 2427024
• DOI: http://doi.org/10.4142/jvs.2018.19.6.759

Abstract

Fowl adenovirus (FAdV) is distributed worldwide and causes economic losses in the poultry industry. The objectives of this study were to determine the hexon and fiber gene changes in an attenuated FAdV isolate from Malaysia in specific pathogen-free chicken embryonated eggs (SPF CEE) and its infectivity in commercial broiler chickens. SPF CEE were inoculated with 0.1 mL FAdV inoculum via the chorioallantoic membrane (CAM) for 20 consecutive passages. The isolate at passage 20 (E20), with a virus titer of 10(8.7)TCIDâ‚…â‚€/mL (TCIDâ‚…â‚€, 50% tissue culture infective dose), was inoculated (0.5 mL) into one-day-old commercial broiler chicks either via oral or intraperitoneal routes. The study demonstrated that 100% embryonic mortality was recorded from E2 to E20 with a delayed pattern at E17 onwards. The lesions were confined to the liver and CAM. Substitutions of amino acids in the L1 loop of hexon at positions 49 and 66, and in the knob of fiber at positions 318 and 322 were recorded in the E20 isolate. The isolate belongs to serotype 8b and is non-pathogenic to broiler chickens, but it is able to induce a FAdV antibody titer. It appears that molecular changes in the L1 loop of hexon and the knob of fiber are markers for FAdV infectivity.

Keyword

amino acids; chickens; chorioallantoic membrane; fowl adenovirus A; specific pathogen-free organisms

MeSH Terms

Adenoviridae*
Amino Acids
Chickens*
Chorioallantoic Membrane
Eggs*
Fowl adenovirus A
Liver
Malaysia*
Mortality
Ovum*
Poultry
Serogroup
Specific Pathogen-Free Organisms
Viral Load
Amino Acids

Figure

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  Fig. 1 Gross lesions of the chorioallantoic membrane (CAM) and liver in chicken embryos following inoculation with fowl adenovirus (FAdV) isolate, UPM1137. (A) CAM of control embryo with a thin and transparent membrane. (B) Normal liver in a control embryo without significant findings. (C) Gross lesions of CAM with thickening and cloudiness in a FAdV-adapted embryo at day 10 post-inoculation (pi) in passage 7 (E7). (D) Diffuse necrosis and petechial hemorrhages of liver (arrow) in a congested embryo at day 10 pi in E7.

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  Fig. 2 Histological lesions of chorioallantoic membrane (CAM) following fowl adenovirus inoculation. (A) Normal CAM in control embryos without significant findings. (B) Severe degeneration, necrosis, and hemorrhages in CAM with numerous basophilic intranuclear inclusion bodies (arrows) in embryos at passage 5 at day 8 post-inoculation. H&E stain (A and B). Scale bars = 50 µm (A), 20 µm (B).

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  Fig. 3 Histological lesions of liver following fowl adenovirus inoculation. (A) Normal liver in control embryos without significant findings. (B) Severe degeneration, necrosis, and hemorrhages in liver with numerous basophilic intranuclear inclusion bodies (arrow) in embryo at passage 20 at day 12 post-inoculation. H&E stain (A and B). Scale bars = 50 µm (A), 20 µm (B).

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  Fig. 4 Electrophoresis of polymerase chain reaction products using primers H1/H2, H3/H4, and FibF/FibR with fragment sizes of (A) 1219 bp, (B) 1319 bp, and (C) 1124 bp amplifying hexon and fiber gene regions of fowl adenovirus (FAdV) in 1% agarose gel from a sample of the original (passage 2 [E2]) and passaged FAdV isolates in specific pathogen-free chicken embryonated eggs (SPF CEE). Lane M, DNA marker 1 kb; Lane 1, positive control (UPM04217); Lane 2, E2 FAdV; Lane 3, E5 FAdV; Lane 4, E10 FAdV; Lane 5, E15 FAdV; Lane 6, E20 FAdV; Lane 7, negative control.

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  Fig. 5 Multiple sequence alignment, obtained via ClustalW [12], of 1166 bp nucleotide sequence of the hexon gene between the original fowl adenovirus (FAdV) isolate (UPM1137E2) and passaged FAdV isolates from passages 5 to 20 (E5–E20) in SPF CEE. The position of the L1 loop region is marked in shaded gray from nucleotide base 6 to base 601 with prominent nt substitutions in the E20 isolate.

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  Fig. 6 Multiple sequence alignment of deduced amino acids in the hexon gene with 388 amino acid residues between the original isolate (UPM1137E2) and passaged fowl adenovirus isolates from passages 5 to 20 (E5–E20) with the L1 loop region (shaded in gray) marked at position 2 to 199. Amino acid substitutions were mainly observed in the variable L1 loop region in the E20 isolate at positions 49 and 66.

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  Fig. 7 Multiple sequence alignment, obtained by ClustalW [12], of a 1094 bp nucleotide sequence of the fiber gene between the original fowl adenovirus (FAdV) isolate (UPM1137E2) and passaged FAdV isolates from passages 5 to 20 (E5–E20) in SPF CEE. The knob region is shaded in darker gray at positions 946 to 1094, a region with a high number of nucleotide base substitutions mainly in the E20 isolate. The tail of the fiber gene is shaded in very light gray and its shaft is shaded in gray.

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  Fig. 8 Multiple sequence alignment, obtained by ClustalW [12], of a 364 deduced amino acid sequence of fiber gene between the original fowl adenovirus (FAdV) isolate (UPM1137E2) and passaged FAdV isolates from passage 5 (E5) (UPM1137E5) to E20 (UPM1137E20) with knob region (shaded in darker gray) marked at positions 316 to 364. The tail of fiber is shaded in very light gray and its shaft is shaded in gray. Amino acid substitutions at position 360 were detected from E5 to E20 with an additional two amino acid changes at positions 318 and 322 in the E20 isolate.

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  Fig. 9 Phylogenetic tree of 198 amino acid residues in the L1 loop of the hexon gene of the fowl adenovirus (FAdV) isolates of this study and of 30 reference FAdV strains retrieved from GenBank. All isolates in SPF CEE from passage 5 (E5) (UPM1137E5) to E20 (UPM1137E20) (shaded in dark gray) were classified as Group E species (shaded in light gray) and were closely related to serotype 8b strains. The early passage isolates E5, E10, and E15 (shaded in dark gray) shared a common ancestor with other Malaysian FAdV strains. The high passage E20 (UPM1137E20) isolate (shaded in dark gray) was closer to the 430-06 and Australian FAdV strains and diverged from the earlier passages isolates. Groups A to E comprised of FAdV strains under serotype 1; serotype 5; serotype 4 and 10; serotype 2, 3, 9, and 11; serotype 6, 7, 8a, and 8b, respectively.

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  Fig. 10 Phylogenetic tree of fiber gene protein of the fowl adenovirus (FAdV) in this study and 30 reference strains retrieved from GenBank based on 364 amino acid residues. Passaged FAdV isolates, passage 5 (E5) (UPM1137E5), E10 (UPM1137E10), E15 (UPM1137E15), and E20 (UPM1137E20) (shaded in dark gray) were classified under group E species (shaded in light gray) and were closely related to serotype 8b strains. Groups A to E comprised of FAdV strains under serotype 1; serotype 5; serotype 4 and 10; serotype 2, 3, 9, and 11; serotype 6, 7, 8a, and 8b, respectively.

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  Fig. 11 Fowl adenovirus (FAdV) antibody response in commercial broiler chickens between Group A (oral inoculation), Group B (intraperitoneal inoculation), and Group C (control) from day 0 to 21 post-inoculation (pi) following inoculation with the attenuated FAdV passage 20 (E20) isolate, UPM1137E20. The antibody titer of FAdV was significantly increased in Groups A and B compared to that in the control group at day 21 pi (*p < 0.05). Bars indicate SEM. ELISA, enzyme-linked immunosorbent assay.

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