Since its emergence in 2009, influenza A(H1N1)pdm09 strain has been one of the seasonal influenza strains and circulated seasonally in humans. Genetic evolution of seasonal influenza viruses is gradual. The close monitoring on the viruses are of note for the vaccine strain recommendations. Since 2009, persistent monitoring of the influenza viruses has been conducted in PKUPH in Beijing, China. We have previously studied the evolution of surface antigen site mutations using the viruses circulated in 2012–2014 [19, 20]. During the 2015–2017 influenza season, influenza A viruses have impacted greatly on the public health (Fig. 1). In order to continuously exploring molecular epidemiological evolution, we analyzed the evolutionary and molecular characteristics of the 33 strain A(H1N1)pdm09 virus strains during the 2015–2017. In consistent with the previous study , the influenza A(H1N1)pdm09 viruses circulated in 2015–2017 in Beijing, China, were belonged to subclade 6B.1A (Fig. 3). The two genetic subclades may be caused by several amino acid substitutions.
Comparing to the HA protein of A/California/07/2009 vaccine strain, A(H1N1)pdm09 viruses circulated in Beijing during 2012–2014 contained several substitutions in the key epitope (Sa, Sb, Ca and Cb) [19, 20], including H155Q in Ca epitopes, L178I/ K180Q in Sa epitope, and S202T in Sb epitope and so on. In this study, all A(H1N1)pdm09 detected during 2015–2017 had S220T, K180Q, S202T mutations, and no H155Q/R, L178I, G187E mutations was detected. The newly discovered S86T was near Cb epitope. The V169T/S181T was near Sa epitope, and G254 was near Ca epitope. They may affect the antigenic properties of the viruses. Substitutions which affect the viral receptor binding profiles have not been detected. It indicated that the evolutions of A(H1N1)pdm09 viruses were gradually in progress. The emergence of unreported mutations, especially those with potential to affect the viral antigen should be of note monitored.
Correspondingly, substitutions in NA antigen epitope have been reported to occur in the A(H1N1)pdm09 viruses during 2012–2014, including NA- K84N, P93S, V106I L139V, I163T, V264I, E287K and S340F. while in this study, NA-V264I mutation occurred in all the A(H1N1)pdm09 viruses detected during 2015–2017. No mutations of NA-K84N, V106I, L139V, I163T, E287K, or S340F has been detected. Moreover, seven locus mutations were newly found, including A86T (1strain), F115L (4 strains), N449T (4 strains), S450V (4 strains), D451T (4 strains), T452L (4 strains), V453W (4 strains).
Comparing with substitutions in the A(H1N1)pdm09 viruses in 2012–2014, several substitutions have not be detected in 2015–2017. It suggested that these variation sites may only be transient variation. On contrast, the increased prevalence of HA-K180Q and NA-V264I mutations from 2012–2014 to 2015–2017 in all strains suggested that several mutations may gradually become dominant over time under the pressure of human immune selection [21, 22].
Many events such as recombination and infection of new hosts can disrupt the balance between HA and NA, but mutations in the HA and NA genes can make up for this imbalance [23,24,25]. Co-occurring mutations, including HA-V169T, A278S, E508G, D518E and NA-V67I were found in the new sites compared with vaccine strains A/California/07/2009 and A/Michigan/45/2015 (H1N1). Symptomatically, sore throat was associated with co-occurring mutations in HA and NA of A(H1N1)pdm09.
While our study highlighted the importance of investigating the effect and mechanism of co-occurring mutation of HA and NA; however, this study had several limitations. First, the study population were all from outpatients who were not severely ill, and the findings may not be generalized to hospitalized or severely ill individuals. Second, the medical data collection was limited, and many important clinical data such as further examination, period of treatment, and prognosis were not available. Third, the number of study patients may not be sufficient to draw the firm conclusions. Fourth, co-occurring mutations we observed had possibility of gradual accumulation of mutations, and we analyzed the co-occurring mutation of HA and NA through the clinical manifestations without mechanism. Therefore, further studies with a larger sample size and more clinical data will be needed to confirm and extend our findings. Evidences of increased fitness or epistasis between the HA and NA segments will also be needed in order to exclude gradual accumulation of mutations.