Abstract
Fowlpox (FP) is an economically important viral disease of commercial poultry. The fowlpox virus (FPV) is primarily characterised by immunoblotting, restriction enzyme analysis in combination with PCR, and/or nucleotide sequencing of amplicons. Whole-genome sequencing (WGS) of FPV directly from clinical specimens prevents the risk of potential genome modifications associated with in vitro culturing of the virus. Only one study has sequenced FPV genomes directly from clinical samples using Nanopore sequencing, however, the study didn’t compare the sequences against Illumina sequencing or laboratory propagated sequences. Here, the suitability of WGS for strain identification of FPV directly from cutaneous tissue was evaluated, using a combination of Illumina and Nanopore sequencing technologies. Sequencing results were compared with the sequence obtained from FPV grown in chorioallantoic membranes (CAMs) of chicken embryos. Complete genome sequence of FPV was obtained directly from affected comb tissue using a map to reference approach. FPV sequence from cutaneous tissue was highly similar to that of the virus grown in CAMs with a nucleotide identity of 99.8%. Detailed polymorphism analysis revealed the presence of a highly comparable number of single nucleotide polymorphisms (SNPs) in the two sequences when compared to the reference genome, providing essentially the same strain identification information. Comparative genome analysis of the map to reference consensus sequences from the two genomes revealed that this field isolate had the highest nucleotide identity of 99.5% with an FPV strain from the USA (Fowlpox virus isolate, FWPV-MN00.2, MH709124) and 98.8% identity with the Australian FPV vaccine strain (FWPV-S, MW142017). Sequencing results showed that WGS directly from cutaneous tissues is not only rapid and cost-effective but also provides essentially the same strain identification information as in-vitro grown virus, thus circumventing in vitro culturing.
Highlights
FP is a common viral disease of commercial poultry and wild birds caused by avipoxviruses [1]
Taxonomic classification of the Nanopore reads from fowlpox virus (FPV)-chorioallantoic membranes (CAMs) by WIMP, considering organisms with more than 100 hits, was as follows: 67%, eukaryotic; 5%, bacterial; and 28%, viral
Whole-genome sequencing (WGS) of a pathogen directly from clinical samples prevents the risk of genome modifications that could occur after passage in vitro [36,37,38]
Summary
FP is a common viral disease of commercial poultry and wild birds caused by avipoxviruses [1]. It is a slow-spreading yet economically important disease, causing a drop in egg production and slow growth of birds followed by mortality [2]. Polypeptide P4b gene, which is highly conserved among all avipoxviruses is mainly targeted for PCR [4,5,6,7,8,9,10]. Based on phylogenetic analysis of the P4b gene, avipoxviruses are divided into three clades and two proposed clades. Two more proposed clades of avipoxviruses include clade D for quail pox virus and clade E for turkey pox virus [11]
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