Whole-genome based strain identification of fowlpox virus directly from cutaneous tissue and propagated virus

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.

Fig 1

Low (A) and high (B) magnification histopathological images of FPV field isolate from comb tissue showing erosion of the epithelial lining in association with necrotic cell debris (N) oedema and mononuclear inflammatory cell infiltration into the dermis (*), hyperplasia and ballooning of the epithelial cells (arrows in A) and round intracytoplasmic eosinophilic inclusion bodies in some of the epithelial cells (arrowheads in B).

Fig 2

Taxonomic classification of FPV-CAMs (A) and FPV-COMB (B) using Kraken2 and visualised by Krona pie chart. Reads identified as homo sapiens are a result of Gallus gallus not being present in the smaller Kraken2 database utilised.

Fig 3. Comparison of near-full length provirus of REV of Australian FPV-COMB and FPV-CAMs with REV strain 104865 (KJ756349).

Fig 4. Genetic relationship of Australian FPV-COMB and FPV-CAMs with reference sequences selected from previous studies.

The relationship was inferred based on phylogenetic analysis of complete genome sequences using the neighbour-joining method with 1000 bootstrap iterations. The labels at branch tips refer to the GenBank accession number followed by the name of the country. Australian FPV-COMB (OK558609) and FPV-CAMs (OK558608) are highlighted in bold.