Department of Veterinary Biosciences, Asia-Pacific Centre for Animal Health, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Werribee, Victoria, Australia.
Department of Veterinary Biosciences, Asia-Pacific Centre for Animal Health, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, Australia.
PLoS One. 2021 Dec 16;16(12):e0261122. doi: 10.1371/journal.pone.0261122. eCollection 2021.
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.
禽痘（FP）是一种对商业家禽具有重要经济意义的病毒性疾病。禽痘病毒（FPV）的主要特征是免疫印迹、与 PCR 相结合的限制性内切酶分析，以及/或扩增子的核苷酸测序。直接从临床标本中对禽痘病毒进行全基因组测序（WGS）可防止与体外培养病毒相关的潜在基因组修饰的风险。只有一项研究使用纳米孔测序直接从临床样本中对禽痘病毒基因组进行了测序，但该研究没有将序列与 Illumina 测序或实验室繁殖的序列进行比较。在这里，使用 Illumina 和纳米孔测序技术的组合，评估了 WGS 直接从皮肤组织中鉴定禽痘病毒株的适用性。测序结果与从鸡胚的鸡胚绒毛尿囊膜（CAM）中生长的 FPV 序列进行了比较。使用映射到参考的方法，直接从受影响的鸡冠组织中获得了 FPV 的完整基因组序列。来自皮肤组织的 FPV 序列与在 CAM 中生长的病毒高度相似，核苷酸同一性为 99.8%。详细的多态性分析表明，与参考基因组相比，两个序列中存在高度可比数量的单核苷酸多态性（SNP），提供了基本相同的菌株鉴定信息。对来自两个基因组的映射到参考共识序列的比较基因组分析表明，该分离株与来自美国的 FPV 株（禽痘病毒分离株，FWPV-MN00.2，MH709124）的核苷酸同一性最高，为 99.5%，与澳大利亚的 FPV 疫苗株（FWPV-S，MW142017）的同一性为 98.8%。测序结果表明，直接从皮肤组织进行 WGS 不仅快速且具有成本效益，而且还提供了与体外生长病毒基本相同的菌株鉴定信息，从而避免了体外培养。
