Comparative analysis of Fowl Aviadenovirus 11 genomes from India in the global context

Introduction

Fowl aviadenoviruses (FAdVs) are non-enveloped, double-stranded DNA viruses belonging to the genus Aviadenovirus within the family Adenoviridae. They are important pathogens of poultry and have been associated with a range of clinical conditions, particularly inclusion body hepatitis (IBH), hepatitis-hydropericardium syndrome (HHS), and, in some cases, respiratory and enteric diseases. Based on molecular and phylogenetic characteristics, FAdVs are currently classified into five species, FAdV-A to FAdV-E, comprising several serotypes (Islam et al., 2026) [5] . Among these, Fowl aviadenovirus D (FAdV-D), corresponds to serotypes FadV-2, 3, 9 and 11. FAdV-11 is of particular interest because of its considerable genetic variability and its association with IBH in chickens (Chavan et al., 2023; Karnam et al., 2026) [1,6] . The FAdV genome is a linear, double-stranded DNA molecule of approximately 43-45 kb and contains several conserved and variable genomic regions. Although the overall genome organization is relatively conserved, substantial genetic variation occurs among FAdV-D isolates (Islam et al., 2026) [5] . The hexon gene, particularly its hypervariable regions, has traditionally been used for molecular classification because the hexon protein is the major capsid protein and contains important antigenic determinants (Franzo et al., 2023, Schachner et al., 2019) [4,10] . However, analyses based solely on partial hexon sequences may not fully represent the genomic relationships among isolates. Other regions, including the fiber genes, penton base, and several non-structural genes, also contribute to genetic differentiation and may provide additional information about the evolutionary history of FadV-D. Genomic diversity in FAdV-D can arise through the accumulation of nucleotide substitutions, insertions and deletions and recombination processes involving different genomic regions. Recombination is particularly important in adenoviruses, as it can generate genomes containing distinct evolutionary histories in different regions (Schachner et al., 2019) [10] . Consequently, isolates showing close relationships based on one gene may exhibit greater divergence when their complete genomes are compared. Whole-genome sequencing and comparative genomic analyses therefore provide a more comprehensive approach for understanding FAdV-D evolution than singlegene analysis alone. The study of FAdV-D genomic diversity is important for understanding virus evolution, epidemiology, geographic dissemination, host adaptation, pathogenicity, and vaccine effectiveness. Characterizing the genetic relationships between contemporary field isolates and previously reported strains can also help identify emerging lineages and conserved genomic regions that may be useful for diagnostic, epidemiological, and vaccine-development purposes. Therefore, comprehensive genomic characterization of the Indian FadV-D, especially FAdV11 strains from India, is essential for elucidating their evolutionary dynamics and for improving surveillance and control strategies in poultry populations.

Materials and Methods

Data set and sequence retrieval

The sequence of three Indian whole genome sequences of FAdV-11 (OR855393, OR192509 and OR194526) were used to retrieve 84 FadV-D global whole genome sequences with more than 98% nucleotide identity from GenBank Nucleotide collection. The complete hexon gene coding sequence of OR194526 FadD-11 stain of India was used retrieve 59 complete hexon gene sequences with more than 98% nucleotide identity and three FAdV2 complete hexon sequences and one recombinant sequence FAdV2/11 hexon sequence from GenBank nucleotide collection.

Sequence alignment and phylogenetic analysis

Sequence alignment and phylogenetic analysis were performed with online Multiple sequence alignment based on fast Fourier transform (MAFFT) online server (https://mafft.cbrc.jp/alignment/server/) (Katoh et al., 2019) [7] . Multiple sequence alignment were performed using Fast Fourier Transform and Neighbor-Joining with two progressive alignment steps (FFT-NS-2) strategy. The phylogenetic trees were constructed using the Jukes-Cantor substitution model with 1,000 bootstrap replicates to assess the reliability of the inferred tree topology.

Phylogenetic tree visualization

The tree was visualized using Phylo.io (https://phylo.io/viewer/) (Robinson et al., 2016) [9] and the phylogenetic tree was further annotated using iTOL (iTOL: Interactive Tree of Life (https://itol.embl.de/) (Letunic et al., 2024) [8] . Th minimum spanning tree visualization was rendered using GrapeTree visualization program (https://achtman-lab.github.io/GrapeTree/MSTree_holder.html) (Zhou et al., 2018) [12] .

Results

Whole genome based phylogenetic analysis

Whole genome based phylogenetic analysis of the three Indian FadV-11 near complete whole genome sequences (OR855393 -43,832 bp, OR192509 -43,631 bp and OR194526 -43,443 bp) with 84 FadV-D global whole genome sequences (ranging from 43,026 bp to 45,193 bp), with more than 98% nucleotide identity and 100% coverage with Indian whole genome sequences, revealed the clustering of the three Indian strains with one Pakistan and one Austrian strain (clade with yellow background in Fig 1). Interestingly, a clade with two strains, one each from Iran and Brazil, shared a common ancestral node with the clade containing the Indian strains revealing a potential shared ancestor with a high bootstrap value. Among these 7 whole genome sequences, the Austrain strain was the earliest to be collected from the field in 2008, followed the Pakistan and Iran strains in 2018, followed by the Indian strains in 2021 and 2022, and then the Brazil strain in 2024. A strain from UK (MK572874 -685 CORR), in the same super clade, one node up from the clade containing the Indian and Iranian strains, is the earliest to be collected from the field approximately in the 1950s to 1960s. The same super clade also has genomes from Ivory coast and Lebanon, in West Africa and West Asia, respectively, both reported collected in 2013. In addition, one strain each from Iran, Brazil, two each from Bulgaria and Taiwan, totaling thirteen sequences along with the above mentioned clade clustered within one super clade node higher-up to the clade containing the Indian strains. The Austrian strain was the earliest to be collected from the field in 2008 within the Indian sub clade, while all other sequences were collected ranging from 2011 to 2024. The other FAdV-11 strains were further separated into many sub clades in the analysis but with poor bootstrap support of 39 (Fig 3). Three Saudi Arabian sequences with the exception of one sequence MK995481, also from Saudi Arabia, clustered together within this major clade. 34 sequences from diverse geographical regions around the world were almost identical and the tree was collapsed for ease of depiction (Fig 3). [2,3,11] . In this study the whole genome sequences and complete hexon gene sequences of FAdV-D from India were compared with global FAdV-D sequences. The phylogenetic tree depiciting 87 whole genome sequences of FAdV (Fig [4] , estimated the mutation rate of the FAdV-Ds in general at 1.55 × 10 -3 subtitutions/site/year based on the comparison of the complete hexon gene sequences available from around the world. However, the estimate is bound to be on the higher side since the hexon gene encodes the protein which is a major neutralizing immune response eliciting target and is under higher selection pressure compared to other segments of the genome of FAdV-D. The study also suggests the clustering of sequences from the Indian subcontinent and a potential spread from Europe to India in the 2000's (Franzo et al., 2023) [4] , corroborating the trend observed in this study with the whole genome seqences. The current study indicates that vaccine strain selection for use in India should be ideally from an Indian isolate due to the variations in the hexon gene. Further, regular surveillance of FAdV-D in India is also warranted to understand the molecular epidemiology of the FAdV-D strains implicated in the pathogenesis of IBH in chickens.

Genetic distance -Minimum spanning tree

Phylogenetic analysis of Indian and global complete hexon gene sequences

References

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