Successful Whole Genome Nanopore Sequencing of Swine Influenza A Virus (swIAV) Directly from Oral Fluids Collected in Polish Pig Herds
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...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-02-01
|
| Series: | Viruses |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1999-4915/15/2/435 |
| _version_ | 1797617808127819776 |
|---|---|
| author | Nick Vereecke Aleksandra Woźniak Marthe Pauwels Sieglinde Coppens Hans Nauwynck Piotr Cybulski Sebastiaan Theuns Tomasz Stadejek |
| author_facet | Nick Vereecke Aleksandra Woźniak Marthe Pauwels Sieglinde Coppens Hans Nauwynck Piotr Cybulski Sebastiaan Theuns Tomasz Stadejek |
| author_sort | Nick Vereecke |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-11T08:01:02Z |
| format | Article |
| id | doaj.art-b8e0673a9ccb4ceab1fe8602bd26d97c |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-11T08:01:02Z |
| publishDate | 2023-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-b8e0673a9ccb4ceab1fe8602bd26d97c2023-11-16T23:48:58ZengMDPI AGViruses1999-49152023-02-0115243510.3390/v15020435Successful Whole Genome Nanopore Sequencing of Swine Influenza A Virus (swIAV) Directly from Oral Fluids Collected in Polish Pig HerdsNick Vereecke0Aleksandra Woźniak1Marthe Pauwels2Sieglinde Coppens3Hans Nauwynck4Piotr Cybulski5Sebastiaan Theuns6Tomasz Stadejek7Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, BelgiumDepartment of Pathology and Veterinary Diagnostic, Institute of Veterinary Medicine, Warsaw University of Life Sciences-SGGW, 02-776 Warsaw, PolandPathoSense BV, 2500 Lier, BelgiumPathoSense BV, 2500 Lier, BelgiumLaboratory of Virology, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, BelgiumGoodvalley Agro S.A., Dworcowa 25, 77-320 Przechlewo, PolandLaboratory of Virology, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, BelgiumDepartment of Pathology and Veterinary Diagnostic, Institute of Veterinary Medicine, Warsaw University of Life Sciences-SGGW, 02-776 Warsaw, PolandInfluenza 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.https://www.mdpi.com/1999-4915/15/2/435epidemiologynanopore sequencingsample storageviral genomicssurveillance |
| spellingShingle | Nick Vereecke Aleksandra Woźniak Marthe Pauwels Sieglinde Coppens Hans Nauwynck Piotr Cybulski Sebastiaan Theuns Tomasz Stadejek Successful Whole Genome Nanopore Sequencing of Swine Influenza A Virus (swIAV) Directly from Oral Fluids Collected in Polish Pig Herds Viruses epidemiology nanopore sequencing sample storage viral genomics surveillance |
| title | Successful Whole Genome Nanopore Sequencing of Swine Influenza A Virus (swIAV) Directly from Oral Fluids Collected in Polish Pig Herds |
| title_full | Successful Whole Genome Nanopore Sequencing of Swine Influenza A Virus (swIAV) Directly from Oral Fluids Collected in Polish Pig Herds |
| title_fullStr | Successful Whole Genome Nanopore Sequencing of Swine Influenza A Virus (swIAV) Directly from Oral Fluids Collected in Polish Pig Herds |
| title_full_unstemmed | Successful Whole Genome Nanopore Sequencing of Swine Influenza A Virus (swIAV) Directly from Oral Fluids Collected in Polish Pig Herds |
| title_short | Successful Whole Genome Nanopore Sequencing of Swine Influenza A Virus (swIAV) Directly from Oral Fluids Collected in Polish Pig Herds |
| title_sort | successful whole genome nanopore sequencing of swine influenza a virus swiav directly from oral fluids collected in polish pig herds |
| topic | epidemiology nanopore sequencing sample storage viral genomics surveillance |
| url | https://www.mdpi.com/1999-4915/15/2/435 |
| work_keys_str_mv | AT nickvereecke successfulwholegenomenanoporesequencingofswineinfluenzaavirusswiavdirectlyfromoralfluidscollectedinpolishpigherds AT aleksandrawozniak successfulwholegenomenanoporesequencingofswineinfluenzaavirusswiavdirectlyfromoralfluidscollectedinpolishpigherds AT marthepauwels successfulwholegenomenanoporesequencingofswineinfluenzaavirusswiavdirectlyfromoralfluidscollectedinpolishpigherds AT sieglindecoppens successfulwholegenomenanoporesequencingofswineinfluenzaavirusswiavdirectlyfromoralfluidscollectedinpolishpigherds AT hansnauwynck successfulwholegenomenanoporesequencingofswineinfluenzaavirusswiavdirectlyfromoralfluidscollectedinpolishpigherds AT piotrcybulski successfulwholegenomenanoporesequencingofswineinfluenzaavirusswiavdirectlyfromoralfluidscollectedinpolishpigherds AT sebastiaantheuns successfulwholegenomenanoporesequencingofswineinfluenzaavirusswiavdirectlyfromoralfluidscollectedinpolishpigherds AT tomaszstadejek successfulwholegenomenanoporesequencingofswineinfluenzaavirusswiavdirectlyfromoralfluidscollectedinpolishpigherds |