Molecular epidemiology and evolution of influenza a and b viruses
Authors:   , Tewawong Nipaporn 
Published: November 2023

The influenza viruses are a major cause of the respiratory severe illness about 3 to 5 million cases and contribute to substantial over 250,000-500,000 death and 200,000 hospitalizations annually worldwide. The influenza vaccination and antiviral prophylaxis are the effective way to prevent the infection and reduce the severity of the disease burden. The aim of the study was to elucidate the epidemiology and molecular evolution of influenza A and B viruses in Thailand between 2010 and 2015. The real time PCR with melting curve analysis for differentiation the lineage specific of influenza B viruses was developed and validated. The lower detection limit was 100 copies per microliter. Using this assay, during 2010 to 2012, there were more B/Vic strains than B/Yam strains (83.5% vs. 16.5%, respectively). B/Vic strains were not detected between February 2013 and the end of 2014. In 2015, B/Yam strains were more prevalent than B/Vic strains (77.5% vs. 22.5%, respectively), while B/Vic dominated the ﬁrst half of 2016 (62.3%). In addition, the whole genome of influenza B viruses was characterized using PCR-sequencing and bioinformatics analysis. The results found 5 inﬂuenza B strains (6.8%) were of mixed lineages between HA and NA genes. Moreover, this study also examined the genetic variability in the nucleotide of encoding HA1 sequences and quantify the antigenic drift of the HA1 domain protein among influenza A(H3N2) and A(H1N1)pdm09 viruses using Pepitope model. The vaccine efficacy for A(H1N1)pdm09 (79.6–93.4%) was generally higher than that of A(H3N2). The emergence of multiple circulating strains of A(H3N2) in 2014-2015 seasons contributed to the reduced vaccine efficacy in Thailand that year. These findings further confirmed the accelerating antigenic drift of the circulating influenza A(H3N2) during this period. Finally, the study was to investigate the molecular evolution of NA gene and examine for the presence of NA substitutions associated with reduced susceptibility to NAIs among seasonal influenza A and B viruses identified in Thailand. The evolutionary patterns of the NA gene indicated a more rapid genetic drift for influenza A than influenza B virus due to higher nucleotide substitution rate, although there was more genetic diversity in influenza B than in influenza A virus. There was an inverse relationship between the evolutionary rate and genealogical diversity. The results found different NA amino acid substitutions at the same residues causing reduced inhibition of influenza A (H3N2) (I222V and S331G) and B  (A395T/D/V and D342S) viruses by NAIs. The recombinant influenza viruses containing these NA mutations were generated using 7+1 reverse genetic system for examining the NA activity and thermostability, NA enzyme inhibition by NAIs, NA enzyme kinetic, genetic stability of NA, and establish influenza A(H3N2) and B virus replication kinetic in vitro. All of recombinant influenza A(H3N2) and B viruses carrying NA substitutions were susceptible to NAIs. There was a difference between wild type and recombinant viruses in NA activity and thermostability. The A/PR8-S331G/R NA viruses revealed increasing of Km and Vmax values, while B/Yam-D342S NA virus contributed to the Km and Vmax values were decreased. The stability of B/Yam-A395V and D342S NA were not stable after three passages in vitro, indicating A395-NA or D342-NA are more suitable in the viral fitness than V395 NA or S342. This finding suggests that NA substitutions of influenza A and B viruses may affect to NA enzyme properties and in vitro virus replication. In conclusion, this study demonstrated the ongoing evolution of genome of influenza A and B viruses, especially HA and NA genes. Continual monitoring of evolutionary dynamics of influenza genome and epidemiological surveillance will assist public health for effective control and prevention influenza infection.