Genome-wide identification of characteristic nucleotide fragments for surveillance and subtype typing of influenza A viruses

Abstract

Objective

Influenza A viruses, members of the Orthomyxoviridae family, are major causative agents of past flu pandemics and can infect a wide range of hosts depending on their hemagglutinin (HA) and neuraminidase (NA) gene combinations. This study aimed to identify genome-wide characteristic nucleotide fragments for rapid detection and subtype typing of influenza A viruses from large-scale genomic data.

Methods

Complete influenza genome sequences were analyzed to identify candidate characteristic fragments specific to influenza A viruses. The fragments were evaluated based on conservation probability, coverage, and specificity across different viral species, subtypes, and hosts. High-coverage fragments were selected for further analysis. Multiplex PCR primers were then designed based on the selected fragments, and their predictive performance was assessed via in silico PCR.

Results

Characteristic fragments from the M gene (20–40 bp) distinguished influenza A viruses with >92 % coverage and >99 % specificity. Forty-four fragments from the HA gene were identified across 26 subtypes, indicating the HA gene's utility for subtype differentiation. Regarding host specificity, canine-derived strains contained unique 20–40 bp fragments, the avian-specific fragment was 20 bp, and no such fragments were detected in other hosts. The designed primers achieved >98 % predicted accuracy for universal detection (M gene) and for H1N1- and H3N2-specific subtypes.

Conclusion

Genome-wide screening of influenza A virus sequences identified highly conserved and subtype-specific nucleotide fragments that enable rapid detection and precise subtyping. These findings provide a valuable resource for molecular surveillance and diagnostic assay development.