Novel HPAIV H5N8 Reassortant (Clade 2.3.4.4b) Detected in Germany

King, Jacqueline; Schulze, Christoph; Engelhardt, A.; Hlinak, Andreas; Lennermann, Sara-Lisa; Rigbers, Kerstin; Skuballa, Jasmin; Staubach, Christoph; Mettenleiter, Thomas C.; Harder, Timm; Beer, Martin; Pohlmann, Anne

doi:10.3390/v12030281

Cited by 44 publications

(49 citation statements)

References 20 publications

(32 reference statements)

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“…The IAV-like subtypes H17N10 and H18N11, described exclusively in bats [59,60], were excluded from the analysis because they do not pose any threats to human or livestock health and are also genetically highly distinct from other IAV subtypes. HPAIV, during early 2020 [61]. Seventy-seven AIV isolates were tested by the SVIP-MPv2 assay alongside the earlier validated SVIP-MPv1 [24] and Spackman et al [16] M-gene RT-qPCRs with an overall close concordance evident among the recorded Cq values, which was also observed between the first two of these assays when applied to the testing of 79 swabs obtained from AIV outbreaks or from experimentally-infected birds (Suppl information 3).…”

Section: Discussionmentioning

confidence: 74%

A universal RT-qPCR assay for “One Health” detection of influenza A viruses

Nagy

Černíková

Kunteová

et al. 2020

Preprint

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AbstractThe mutual dependence of human and animal health is central to the One Health initiative as an integrated strategy for infectious disease control and management. A crucial element of the One Health includes preparation and response to influenza A virus (IAV) threats at the human-animal interface. The IAVs are characterized by extensive genetic variability, they circulate among different hosts and establish host-specific lineages. The four main host reservoirs are: avian, swine, human and equine, with occasional transmission to other mammalian species. The host diversity is mirrored in the range of the RT-qPCR assays for IAV detection. Different assays are recommended by the responsible health authorities for generic IAV detection in birds, swine or humans. In order to unify IAV monitoring in different hosts and apply the One Health approach, we developed a single RT-qPCR assay for universal detection of all IAVs of all subtypes, species origin and global distribution. The assay design was centred on a highly conserved region of the IAV MP-segment identified by a comprehensive analysis of 99,353 sequences. The reaction parameters were effectively optimised with efficiency of 93-97% and LOD95% of approximately ten IAV templates per reaction. The assay showed high repeatability, reproducibility and robustness. The extensive in silico evaluation demonstrated high inclusivity, i.e. perfect sequence match in the primers and probe binding regions, established as 94.6% for swine, 98.2% for avian and 100% for human H3N2, pandemic H1N1, as well as other IAV strains, resulting in an overall predicted detection rate of 99% on the analysed dataset. The theoretical predictions were confirmed and extensively validated by collaboration between six veterinary or human diagnostic laboratories on a total of 1970 specimens, of which 1455 were clinical and included a diverse panel of IAV strains.

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Section: Discussionmentioning

confidence: 74%

A universal RT-qPCR assay for “One Health” detection of influenza A viruses

Nagy

Černíková

Kunteová

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…Validation included testing AIVs of diverse origins which included all 16 HA and nine NA subtypes. The AIVs included isolates and clinical specimens obtained from the most recent incursion of the important GsGd lineage into Europe, namely the H5N8 HPAIV (clade 2.3.4.4b), during early 2020 [ 61 ]. Seventy-seven AIV isolates were tested by the SVIP-MPv2 assay alongside the earlier validated SVIP-MPv1 [ 24 ] and Spackman et al [ 16 ] MP-gene RT-qPCRs with an overall close concordance evident among the recorded Cq values, which was also observed between the first two of these assays when applied to the testing of 79 swabs obtained from AIV outbreaks or from experimentally-infected birds (S3).…”

Section: Discussionmentioning

confidence: 99%

A universal RT-qPCR assay for “One Health” detection of influenza A viruses

et al. 2021

View full text Add to dashboard Cite

The mutual dependence of human and animal health is central to the One Health initiative as an integrated strategy for infectious disease control and management. A crucial element of the One Health includes preparation and response to influenza A virus (IAV) threats at the human-animal interface. The IAVs are characterized by extensive genetic variability, they circulate among different hosts and can establish host-specific lineages. The four main hosts are: avian, swine, human and equine, with occasional transmission to other mammalian species. The host diversity is mirrored in the range of the RT-qPCR assays for IAV detection. Different assays are recommended by the responsible health authorities for generic IAV detection in birds, swine or humans. In order to unify IAV monitoring in different hosts and apply the One Health approach, we developed a single RT-qPCR assay for universal detection of all IAVs of all subtypes, species origin and global distribution. The assay design was centred on a highly conserved region of the IAV matrix protein (MP)-segment identified by a comprehensive analysis of 99,353 sequences. The reaction parameters were effectively optimised with efficiency of 93–97% and LOD95% of approximately ten IAV templates per reaction. The assay showed high repeatability, reproducibility and robustness. The extensive in silico evaluation demonstrated high inclusivity, i.e. perfect sequence match in the primers and probe binding regions, established as 94.6% for swine, 98.2% for avian and 100% for human H3N2, pandemic H1N1, as well as other IAV strains, resulting in an overall predicted detection rate of 99% on the analysed dataset. The theoretical predictions were confirmed and extensively validated by collaboration between six veterinary or human diagnostic laboratories on a total of 1970 specimens, of which 1455 were clinical and included a diverse panel of IAV strains.

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“…The MinION proved to be adaptably applicable for not only avian and human isolates, but also for representative clinical swab samples. In addition, swab samples from the recent 2020 outbreak of a novel IAV clade 2.3.4.4b H5N8 reassortant in Germany were successfully sequenced to produce full genome sequences using this method [25], demonstrating the practicality and applicability of the respective workflow.…”

Section: Discussionmentioning

confidence: 99%

Rapid multiplex MinION nanopore sequencing workflow for Influenza A viruses

et al. 2020

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Background: Due to the frequent reassortment and zoonotic potential of influenza A viruses, rapid gain of sequence information is crucial. Alongside established next-generation sequencing protocols, the MinION sequencing device (Oxford Nanopore Technologies) has become a serious competitor for routine whole-genome sequencing. Here, we established a novel, rapid and high-throughput MinION multiplexing workflow based on a universal RT-PCR. Methods: Twelve representative influenza A virus samples of multiple subtypes were universally amplified in a onestep RT-PCR and subsequently sequenced on the MinION instrument in conjunction with a barcoding library preparation kit from the rapid family and the MinIT performing live base-calling. The identical PCR products were sequenced on an IonTorrent platform and, after final consensus assembly, all data was compared for validation. To prove the practicability of the MinION-MinIT method in human and veterinary diagnostics, we sequenced recent and historical influenza strains for further benchmarking. Results: The MinION-MinIT combination generated over two million reads for twelve samples in a six-hour sequencing run, from which a total of 72% classified as quality screened, trimmed and mapped influenza reads to produce full genome sequences. Identities between the datasets of > 99.9% were achieved, with 100% coverage of all segments alongside a sufficient confidence and 4492fold mean depth. From RNA extraction to finished sequences, only 14 h were required. Conclusions: Overall, we developed and validated a novel and rapid multiplex workflow for influenza A virus sequencing. This protocol suits both clinical and academic settings, aiding in real time diagnostics and passive surveillance.

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Novel HPAIV H5N8 Reassortant (Clade 2.3.4.4b) Detected in Germany

Cited by 44 publications

References 20 publications

A universal RT-qPCR assay for “One Health” detection of influenza A viruses

A universal RT-qPCR assay for “One Health” detection of influenza A viruses

A universal RT-qPCR assay for “One Health” detection of influenza A viruses

Rapid multiplex MinION nanopore sequencing workflow for Influenza A viruses

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