Charged amino acid variability related to N-glyco -sylation and epitopes in A/H3N2 influenza: Hem -agglutinin and neuraminidase

Background
The A/H3N2 influenza viruses circulated in humans have been shown to undergo antigenic drift, a process in which amino acid mutations result from nucleotide substitutions. There are few reports regarding the charged amino acid mutations. The purpose of this paper is to explore the relations between charged amino acids, N-glycosylation and epitopes in hemagglutinin (HA) and neuraminidase (NA).
Methods
A total of 700 HA genes (691 NA genes) of A/H3N2 viruses were chronologically analyzed for the mutational variants in amino acid features, N-glycosylation sites and epitopes since its emergence in 1968.
Results
It was found that both the number of HA N-glycosylation sites and the electric charge of HA increased gradually up to 2016. The charges of HA and HA1 increased respectively 1.54-fold (+7.0 /+17.8) and 1.08-fold (+8.0/+16.6) and the number of NGS in nearly doubled (7/12). As great diversities occurred in 1990s, involving Epitope A, B and D mutations, the charged amino acids in Epitopes A, B, C and D in HA1 mutated at a high frequency in global circulating strains last decade. The charged amino acid mutations in Epitopes A (T135K) has shown high mutability in strains near years, resulting in a decrease of NGT135-135. Both K158N and K160T not only involved mutations charged in epitope B, but also caused a gain of NYT158-160. Epitope B and its adjacent N-glycosylation site NYT158-160 mutated more frequently, which might be under greater immune pressure than the rest.
Conclusions
The charged amino acid mutations in A/H3N2 Influenza play a significant role in virus evolution, which might cause an important public health issue. Variability related to both the epitopes (A and B) and N-glycosylation is beneficial for understanding the evolutionary mechanisms, disease pathogenesis and vaccine research.

背景
已有研究表明，在人群中循环传播的甲型H3N2流感病毒（Influenza A/H3N2）会发生抗原漂移（antigenic drift），该过程由核苷酸替换引发氨基酸突变。目前针对带电氨基酸突变的相关报道较为匮乏。本研究旨在探讨血凝素（hemagglutinin, HA）与神经氨酸酶（neuraminidase, NA）中带电氨基酸、N-糖基化与抗原表位之间的关联。
方法
自1968年甲型H3N2病毒出现以来，本研究按时间序列对700条HA基因（对应691条NA基因）的氨基酸特征突变体、N-糖基化位点及抗原表位进行了分析。
结果
研究发现，截至2016年，HA的N-糖基化位点数量与HA的净电荷均呈逐渐上升趋势。其中HA与HA1的电荷分别提升了1.54倍（+7.0/+17.8）与1.08倍（+8.0/+16.6），而N-糖基化位点（N-glycosylation sites, NGS）的数量几乎翻倍（7/12）。20世纪90年代出现了显著的病毒多样性，涉及表位A、B及D的突变；近十年间，全球流行毒株中HA1的表位A、B、C及D区域的带电氨基酸发生高频突变。表位A中的带电氨基酸突变（T135K）在近年毒株中表现出较高的突变率，导致NGT135-135位点消失。K158N与K160T两类突变不仅涉及表位B的带电氨基酸改变，还使NYT158-160位点获得了N-糖基化修饰。表位B及其相邻的N-糖基化位点NYT158-160的突变频率更高，提示其可能承受了相较于其他区域更强的免疫压力。
结论
甲型H3N2流感病毒中的带电氨基酸突变在病毒进化过程中发挥着重要作用，可能会引发重大公共卫生问题。与抗原表位（A、B型）及N-糖基化相关的病毒变异，有助于深入理解病毒的进化机制、疾病发病机制以及疫苗研发工作。