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

Abstract

<jats:title>Abstract</jats:title><jats:p>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 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 LOD<jats:sub>95%</jats:sub> of approximately ten IAV templates per reaction. The assay showed high repeatability, reproducibility and robustness. The extensive <jats:italic>in silico</jats:italic> 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.</jats:p>

Emergence and spread of novel H5N8, H5N5 and H5N1 clade 2.3.4.4 highly pathogenic avian influenza in 2020

“…Samples submitted by the National Veterinary Reference Centre, Kazakhstan to the Animal and Plant Health Agency, UK on FTA cards were triaged diagnostically for viral nucleic acid. All samples were positive for H5N8 nucleic acid by subtype-specific real-time RT-PCR [ 4 , 5 ]. Genetic analyses of the whole genomes from the Kazakhstan outbreaks demonstrated that for all segments they were highly similar to the H5N8 virus from Iraq as evidenced by relatively short branch lengths within this Iraqi-like cluster linked by a long branch to ancestral strains ( Figure 1 (B)).…”

Genotype Diversity, Wild Bird-to-Poultry Transmissions, and Farm-to-Farm Carryover during the Spread of the Highly Pathogenic Avian Influenza H5N1 in the Czech Republic in 2021/2022

“…Total nucleic acid was extracted from 200µl supernatants of pooled organs or swabs (MagNAPure Compact, MagNAPure 24 or MagNAPure 96 instruments, Roche) and eluted into 50µl. For detection and identification of H5N1, RT-qPCR methods specific for generic influenza A virus and H5 and N1 subtypes were performed in combination with cleavage site sequencing [15,16,17,18].…”

Genotype Uniformity, Wild Bird-to-Poultry Transmissions, and Farm-to-Farm Carryover during the Spread of Highly Pathogenic Avian Influenza H5N8 in the Czech Republic in 2021

“…Total nucleic acid was extracted from 200µl of supernatant of pooled organs and swabs (MagNAPure Compact, MagNAPure 24 or MagNAPure 96 instruments, Roche) and eluted to 50µl. For H5N8 HPAI detection and identification, generic influenza A virus, H5 and N8 subtype specific RT-qPCR methods in combination with cleavage site sequencing [5][6][7] were performed.…”