![]() The low-fidelity, “error-prone” RNA-dependent RNA polymerase (RdRp) of IAVs lacks the 3’ to 5’ exonuclease proofreading capability, leading to a rapid mutation rate that ranges from 0.4 × 10 −3 to 2.0 × 10 −6 mutations per nucleotide per year, depending on virus strain and gene. Evolution and antigenic variations of influenza viruses In addition, several novel accessory proteins of IAVs were identified that modulate viral pathogenicity, such as PB1-F2 and PB1-N40 encoded by the PB1 gene and PA-X, PA-N155, and PA-N182 by the PA gene. Segments 1 (PB2), 2 (PB1), 3 (PA), 4 (HA), 5 (NP), 6 (NA), 7 (MP), and 8 (NS) of IAVs and IBVs encode polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), polymerase acidic protein (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix proteins (M1 and M2), and nonstructural proteins (NS1 and NS2), respectively, which will be described in the subsequent sections. Influenza A viruses (IAVs) and influenza B viruses (IBVs) contain 8 viral RNA (vRNA) gene segments, whereas influenza C viruses (ICVs) and influenza D viruses (IDVs) contain 7 vRNA gene segments. Influenza viruses contain segmented, negative-sense, single-stranded RNA genomes. Influenza viruses belong to the Orthomyxoviridae family and are classified into four genera including type A, B, C, and the emerging type D based on their antigenic differences in the nucleoprotein and matrix 1 protein. ![]() Unpredictably, but less frequently, global influenza pandemics occur, infecting 20–40% of the population in a single year and dramatically raising death rates above normal levels. Seasonal influenza infections are associated with ~290,000–650,000 deaths annually worldwide, which includes ~12,000–61,000 deaths each year in the United States (US) alone. ![]() The influenza virus is a recurring threat to public health. In this study, we review the primary analytic methods used for antigenic characterization of influenza and SARS-CoV-2 and discuss the barriers of these methods and current developments for addressing these challenges. SARS-CoV-2 characterization has faced similar challenges with the additional barrier of the need for facilities with high biosafety levels due to its infectious nature. ![]() For influenza, these barriers include the requirement for a large virus quantity to perform the assays, more than what can typically be provided by the clinical samples alone, cell- or egg-adapted mutations that can cause antigenic mismatch between the vaccine strain and circulating viruses, and up to a 6-month duration of vaccine development after vaccine strain selection, which allows viruses to continue evolving with potential for antigenic drift and, thus, antigenic mismatch between the vaccine strain and the emerging epidemic strain. While great strides have been made for evaluating the antigenic properties of these viruses, multiple challenges prevent efficient vaccine strain selection and accurate assessment. These techniques have been improved upon over time for increased analytical capacity, and some have been mobilized for the rapid characterization of the SARS-CoV-2 virus as well as its variants, facilitating the development of highly effective vaccines within 1 year of the initially reported outbreak. Primary analytic methods, including enzyme-linked immunosorbent/lectin assays, hemagglutination inhibition, neuraminidase inhibition, micro-neutralization assays, and antigenic cartography, have been widely used in the field of influenza research. Antigenic characterization of emerging and re-emerging viruses is necessary for the prevention of and response to outbreaks, evaluation of infection mechanisms, understanding of virus evolution, and selection of strains for vaccine development.
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