Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium.
PathoSense BV, 2500 Lier, Belgium.
Viruses. 2023 Feb 4;15(2):435. doi: 10.3390/v15020435.
Influenza A virus (IAV) is a single-stranded, negative-sense RNA virus and a common cause of seasonal flu in humans. Its genome comprises eight RNA segments that facilitate reassortment, resulting in a great variety of IAV strains. To study these processes, the genetic code of each segment should be unraveled. Fortunately, new third-generation sequencing approaches allow for cost-efficient sequencing of IAV segments. Sequencing success depends on various factors, including proper sample storage and processing. Hence, this work focused on the effect of storage of oral fluids and swIAV sequencing. Oral fluids (n = 13) from 2017 were stored at -22 °C and later transferred to -80 °C. Other samples (n = 21) were immediately stored at -80 °C. A reverse transcription quantitative PCR (RT-qPCR) pre- and post-storage was conducted to assess IAV viral loads. Next, samples were subjected to two IAV long-read nanopore sequencing methods to evaluate success in this complex matrix. A significant storage-associated loss of swIAV loads was observed. Still, a total of 17 complete and 6 near-complete Polish swIAV genomes were obtained. Genotype T, (H1avN2, seven herds), P (H1N1pdm09, two herds), U (H1avN1, three herds), and A (H1avN1, 1 herd) were circulated on Polish farms. In conclusion, oral fluids can be used for long-read swIAV sequencing when considering appropriate storage and segment amplification protocols, which allows us to monitor swIAV in an animal-friendly and cost-efficient manner.
甲型流感病毒（IAV）是一种单链、负义 RNA 病毒，是人类季节性流感的常见病因。其基因组由 8 个 RNA 片段组成，这些片段促进了重配，导致了大量的 IAV 株。为了研究这些过程，每个片段的遗传密码都应该被解开。幸运的是，新的第三代测序方法可以实现 IAV 片段的成本效益测序。测序的成功取决于各种因素，包括适当的样本储存和处理。因此，这项工作集中研究了储存口腔液和 swIAV 测序的影响。2017 年的 13 份口腔液样本储存在-22°C，随后转移到-80°C。其他 21 份样本立即储存在-80°C。在储存前后进行逆转录定量 PCR（RT-qPCR）以评估 IAV 病毒载量。然后，对样本进行两种 IAV 长读长纳米孔测序方法，以评估在这种复杂基质中的成功。观察到与储存相关的 swIAV 载量显著损失。尽管如此，总共获得了 17 个完整和 6 个接近完整的波兰 swIAV 基因组。在波兰农场流行的基因型为 T（H1avN2，七个畜群）、P（H1N1pdm09，两个畜群）、U（H1avN1，三个畜群）和 A（H1avN1，一个畜群）。总之，考虑到适当的储存和片段扩增方案，口腔液可用于长读 swIAV 测序，这使得我们能够以动物友好和具有成本效益的方式监测 swIAV。
