不同宿主H1N1病毒血凝素蛋白(HA)受体结合位点的变异特征

2009年全球性爆发的H1N1病毒已经导致213个国家和地区受到感染, 有16 226人死亡。病毒与宿主细胞表面受体的结合是病毒感染不可缺少的第一步, 从而导致病毒膜与宿主细胞膜的融合。血凝素(Hemagglutinin, HA)就是介导这种受体结合与膜融合的病毒蛋白, 受体结合位点(Receptor binding sites, RBSs)位于HA蛋白三聚体中每个单体的球形头部, 主要由190位螺旋(190~198aa)、130位环(135~138aa)和220位环(221~228)3个二级结构域组成。文章收集了1918~2009年间1 221株H1N1病毒株的HA1序列(长度为327个氨基酸残基), 通过序列比对、各位点氨基酸残基的熵值以及3D结构模拟等生物信息学研究。结果显示不同宿主的不同病毒RBSs具有不同的熵值, 而且不同宿主的病毒HA1其RBSs具有不同的优势序列。3D结构模拟也显示了H1N1不同HA1之间在190位螺旋构象上的细微差异。该研究揭示了不同HA1上RBSs的一些新的特征, 为进一步探讨病毒感染的机理提供了新的信息     

甲型H1N1流感病毒HA蛋白结构和功能研究进展

自2009年3月,甲型H1N1流感疫情相继在包括我国在内的许多国家暴发,对人体健康和社会经济发展造成了严重危害。血凝素（HA）蛋白是重要的病毒表面糖蛋白,主要有3种功能：①与宿主细胞表面受体结合;②引起病毒包膜与靶细胞间的膜融合;③刺激机体产生中和性抗体。本文综合了近年来的研究成果,对甲型H1N1流感病毒HA蛋白结构、主要功能、进化、抗原性的研究进展进行了综述。     

甲型H1N1流感病毒NA、PB2和PA表位抗原区段的克隆和表达

目的:分析比对甲型H1N1流感病毒神经氨酸酶(NA)、聚合酶B2(PB2)和聚合酶A(PA)抗原的序列,克隆表达保守的和变异的表位抗原区段,为免疫学诊断试剂的研究提供候选抗原。方法:采用BioSun生物学软件比较新近公布的A/H1N1流感病毒和我国猪流感病毒浙江株NA、PB2和PA抗原序列,并预测筛选NA、PB2和PA抗原表位保守区段与变异区段,采用PCR逐步合成法合成NA、PB2和PA表位抗原区段基因序列,并利用原核表达载体pBVIL1进行克隆表达。结果:筛选的保守表位抗原区段和变异表位抗原区段为NA/135～180aa、NA/310～350aa、PB2/1～80aa、PA/41～90aa、PA/231～280aa和PA/341～400aa;获得6条表位抗原区段基因序列,且6条表位抗原区段均获得了高效表达,得到纯化后的抗原。结论:获得了A/H1N1流感病毒NA、PB2和PA表位抗原区段,为进一步研制特异的甲型流感病毒快速诊断试剂提供了抗原储备。     

流感病毒NP表位序列增强H7N9 HA DNA疫苗免疫效果研究

为增强H7N9流感病毒HA DNA疫苗的免疫效果,本文构建了含有流感病毒NP的5个重复优势T表位或B表位(包括B_1线性表位和B_2构象表位)的表达质粒(简称NPT和NPB),以小鼠为动物模型,将NPT和NPB分别与H7N9流感病毒HA DNA混合免疫BALB/c小鼠1次,21 d后用致死剂量(20 LD_(50))的H7N9流感病毒攻击小鼠。通过检测免疫后小鼠血清特异性抗体滴度和攻毒后的免疫保护性指标-存活率、肺部残余病毒滴度及体重丢失率,评价表位质粒与HA DNA疫苗混合免疫对小鼠的保护效果。试验结果表明:用致死性H7N9流感病毒攻击小鼠后,HA DNA组小鼠全部死亡,HA+NPT免疫组小鼠存活率达到100%,HA+NPB1和HA+NPB2免疫组,小鼠存活率分别为30%和75%;混合免疫HA+NPT后小鼠抗体滴度升高明显,攻毒后小鼠体重丢失明显降低。综上表明,含有5个NP优势T表位或B表位的表达质粒能增强H7N9流感病毒HA DNA疫苗的免疫效果,混合免疫一次可以使小鼠得到较好的保护。HA DNA和表位疫苗的混合免疫,节约了疫苗用量,降低了免疫成本,为流感病毒核酸疫苗的临床开发提供了一定的试验基础。     

一种含不同流感病毒血凝素抗原表位的核酸疫苗

流感病毒表面抗原血凝素( hemagglutinin,HA)是流感核酸疫苗重要的靶抗原,针对HA的保护性中和抗体主要由HA上的五个抗原表位诱导产生.在本文中,我们构建了一种以新甲型H1N1流感病毒HA1为骨架的含2个A/PR/8( H1N1)流感病毒HA抗原表位和3个新甲型H1N1流感病毒HA抗原表位的核酸疫苗,并在B...     

甲型H1N1流感病毒血凝素蛋白单抗杂交瘤细胞株的制备与初步鉴定

目的:建立具有高特异、高效价的甲型H1N1流感病毒血凝素蛋白(HA)单抗的杂交瘤细胞株。方法:以纯化的昆虫杆状病毒表达的甲型H1N1流感病毒HA蛋白为免疫原免疫BALB/c小鼠,取脾细胞与Sp2/0小鼠骨髓瘤细胞融合,通过有限稀释法筛选阳性克隆,经ELISA和Western blot分析单抗的特性和特异性。结果:获得6株甲型H1N1流感HA抗原特异单克隆抗体杂交瘤细胞株,抗原肽库ELISA检测结果表明其中3株(1E12,3F12,1C11)单抗只与甲型H1N1流感HA抗原肽库反应,不与H5N1病毒HA抗原肽库反应;Western blot分析表明,单抗1B3只特异识别甲型H1N1流感HA抗原,而与其他季节性甲流病毒(H1,H3)及人禽流感H5N1病毒不反应。结论:所获杂交瘤细胞株特异性强,效价高,分泌抗体性能稳定,为分析甲型H1N1流感病毒抗原性位点、建立诊断试剂奠定了基础。     

甲型H1N1流感病毒HA基因RT-LAMP检测方法的建立与初步应用

目的:建立反转录环介导等温扩增技术(RT-LAMP)检测甲型H1N1流感病毒HA基因的方法,并用于检测临床样本.方法:通过在线软件Primer Explorer V4设计H1N1 HA基因的RT-LAMP引物,建立RT-LAMP检测方法,并评价其灵敏度和特异度.结果:与传统RT-PCR方法相比,RT-LAMP检测方法具备更高的灵敏度,达到10个拷贝,并且具有良好的特异性;在76份呼吸道感染儿童的咽拭子标本中检测到2份阳性,与RT-PCR方法检测结果相同.结论:建立的H1N1 RT-LAMP检测方法灵敏特异,简单快速,具备H1N1现场检测应用前景.     

云南省2009～2014年甲型H1N1流感病毒HA及NA基因特征分析

了解云南省2009~2014年甲型H1N1流感病毒的流行趋势,研究HA和NA基因进化特征。对云南省近6年来上报的流感监测病例数据进行病原谱总结,挑选出23株甲型H1N1流感毒株进行HA及NA基因分析。利用MEGA 5.0软件对测序结果构建进化树分析基因同源性。2009~2014年云南省共监测到4次甲型H1N1流感流行高峰,核酸检测结果中甲型H1N1流感占检出总量的28.8%。测序结果显示,HA与NA基因均分为3个类群,检测到一株具有H275Y突变位点的毒株。甲型H1N1流感是导致本省流感流行的重要亚型之一,2009~2014年间分离的毒株主要有Goup1、Gourp7和Gourp6三个支系,绝大部分甲型H1N1流感毒株仍对神经氨酸酶抑制剂敏感。     

H5N1亚型AIVHA抗原表位的高效表达及其抗原活性分析

血凝素(hemagglutinin,HA)蛋白是禽流感病毒(avian influenza virus,AIV)的一个重要表面抗原性蛋白,在疾病诊断和防治上有重要意义。本研究为了探讨一种更为简便有效的HA重组蛋白表达途径,利用生物信息学软件,对H5N1亚型AIVHA基因编码的氨基酸序列进行分析,在分析其在大肠杆菌中的密码子偏好性、稀有密码子分布情况及有关蛋白的抗原性等重要特性后,构建了HA抗原表位重组表达质粒pET-32a(+)-HA。经测试,该重组质粒在1mmol/LIPTG诱导剂作用下诱导过夜,能在大肠杆菌Rosetta-gami B(DE3)中高效表达,并得到48.1kD大小的目的重组表达蛋白。重组蛋白用6×His-tagged protein纯化试剂盒纯化后,与福氏佐剂等量混合制备成抗原,以200μg/鸡的剂量皮下注射2月龄SPF鸡3次,采血分离血清。Western-Blot试验结果表明,该重组表达蛋白能分别与所制备的高免鸡血清及H5N1亚型AIV阳性血清发生特异性反应,在硝酸纤维素膜上出现特异性杂交带。说明本试验研究的HA抗原重组表达蛋白具有良好的免疫原性和反应原性,保留了HA蛋白的抗原活性,提示该重组蛋白在H5亚型AIV的防治技术研究中具有重要的实际应用价值。     

Antibody Recognition of the Pandemic H1N1 Influenza Virus Hemagglutinin Receptor Binding Site

Influenza virus is a global health concern due to its unpredictable pandemic potential. This potential threat was realized in 2009 when an H1N1 virus emerged that resembled the 1918 virus in antigenicity but fortunately was not nearly as deadly. 5J8 is a human antibody that potently neutralizes a broad spectrum of H1N1 viruses, including the 1918 and 2009 pandemic viruses. Here, we present the crystal structure of 5J8 Fab in complex with a bacterially expressed and refolded globular head domain from the hemagglutinin (HA) of the A/California/07/2009 (H1N1) pandemic virus. 5J8 recognizes a conserved epitope in and around the receptor binding site (RBS), and its HCDR3 closely mimics interactions of the sialic acid receptor. Electron microscopy (EM) reconstructions of 5J8 Fab in complex with an HA trimer from a 1986 H1 strain and with an engineered stabilized HA trimer from the 2009 H1 pandemic virus showed a similar mode of binding. As for other characterized RBS-targeted antibodies, 5J8 uses avidity to extend its breadth and affinity against divergent H1 strains. 5J8 selectively interacts with HA insertion residue 133a, which is conserved in pandemic H1 strains and has precluded binding of other RBS-targeted antibodies. Thus, the RBS of divergent HAs is targeted by 5J8 and adds to the growing arsenal of common recognition motifs for design of therapeutics and vaccines. Moreover, consistent with previous studies, the bacterially expressed H1 HA properly refolds, retaining its antigenic structure, and presents a low-cost and rapid alternative for engineering and manufacturing candidate flu vaccines.     

Structure and Receptor Binding Specificity of Hemagglutinin H13 from Avian Influenza A Virus H13N6

Interspecies transmission (host switching/jumping) of influenza viruses is a key scientific question that must be addressed. In addition to the vigorous research on highly pathogenic avian influenza viruses (HPAIVs), studies of the mechanism of interspecies transmission of low-pathogenic avian influenza viruses (LPAIVs) could also provide insights into host tropism and virulence evolution. Influenza A viruses harboring hemagglutinin (HA) H13 (e.g., H13N6) are LPAIVs. In this study, soluble H13 HA glycoprotein was purified, and its receptor binding activity was characterized. The results revealed that H13 exclusively binds the avian α2-3-linked sialic acid receptor; no binding to the mammalian α2-6-linked sialic acid receptor was detected. Furthermore, the molecular basis of the H13 receptor binding specificity was revealed by comparative analysis of the crystal structures of both receptor-bound H13 and H5 HAs, which might be contributed by the hydrophobic residue V186. Work with an H13N186 mutant confirmed the importance of V186 in the receptor binding specificity of H13 HA, which shows that the mutant protein reduced the binding of an avian receptor analog but increased the binding of a human receptor analog. Detailed structural analysis also demonstrated that the conserved binding sites of the recently well-studied broadly neutralizing human monoclonal antibodies targeting the HA2 domain are found in H13. Our results expand our understanding of virulence evolution, receptor binding preference, and species tropism of the LPAIVs and HPAIVs.     

不同特征描述下H1N1病毒血凝素蛋白序列的比较分析

在传统表征蛋白质序列的40维特征向量的基础上,依据氨基酸的种类与理化性质,将蛋白质序列40维向量分解为20维、4维和16维3种子特征向量描述。结合33条H1N1流感病毒血凝素(hemagglutinin,HA)蛋白质序列和统计学相关性分析理论,进行了蛋白质序列两两之间及每条病毒蛋白质序列对应的不同子特征向量之间的相关性分析,发现病毒蛋白质序列之间存在高度相关性,且每条病毒蛋白质序列对应的20维子特征向量与其他两种子特征向量之间均不显著相关,而4维与16维子特征向量之间显著相关。进一步依据不同的特征向量对33条HA蛋白质序列进行分类,研究发现依据40维特征向量与16维特征向量进行的分类结果高度一致。因此,在不影响表征病毒序列特性的前提下,对于已有的表征蛋白质序列的40维特征向量,可以用16维的特征向量进行代替,以减少计算复杂度。     

Virulence-Associated Substitution D222G in the Hemagglutinin of 2009 Pandemic Influenza A(H1N1) Virus Affects Receptor Binding

The clinical impact of the 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively low. However, amino acid substitution D222G in the hemagglutinin of pdmH1N1 has been associated with cases of severe disease and fatalities. D222G was introduced in a prototype pdmH1N1 by reverse genetics, and the effect on virus receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models was investigated. pdmH1N1 with D222G caused ocular disease in mice without further indications of enhanced virulence in mice and ferrets. pdmH1N1 with D222G retained transmissibility via aerosols or respiratory droplets in ferrets and guinea pigs. The virus displayed changes in attachment to human respiratory tissues in vitro, in particular increased binding to macrophages and type II pneumocytes in the alveoli and to tracheal and bronchial submucosal glands. Virus attachment studies further indicated that pdmH1N1 with D222G acquired dual receptor specificity for complex α2,3- and α2,6-linked sialic acids. Molecular dynamics modeling of the hemagglutinin structure provided an explanation for the retention of α2,6 binding. Altered receptor specificity of the virus with D222G thus affected interaction with cells of the human lower respiratory tract, possibly explaining the observed association with enhanced disease in humans.In April 2009, the H1N1 influenza A virus of swine origin was detected in humans in North America (9, 12, 42). Evidence for its origin came from analyses of the viral genome, with six gene segments displaying the closest resemblance to American “triple-reassortant” swine viruses and two to “Eurasian-lineage” swine viruses (13, 42). After this first detection in humans, the virus spread rapidly around the globe, starting the first influenza pandemic of the 21st century. The 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively mild, with a spectrum of disease ranging from subclinical infections or mild upper respiratory tract illness to sporadic cases of severe pneumonia and acute respiratory distress syndrome (3, 11, 27, 29, 30, 37). Overall, the case-fatality rate during the start of the pandemic was not significantly higher than in seasonal epidemics in most countries. However, a marked difference was observed in the case-fatality rate in specific age groups, with seasonal influenza generally causing highest mortality in elderly and immunocompromised individuals, and the pdmH1N1 affecting a relatively large proportion of (previously healthy) young individuals (3, 11, 27, 29, 30, 37).Determinants of influenza A virus virulence have been mapped for a wide variety of zoonotic and pandemic influenza viruses to the polymerase genes, hemagglutinin (HA), neuraminidase (NA), and nonstructural protein 1 (NS1). Such virulence-associated substitutions generally facilitate more efficient replication in humans via improved interactions with host cell factors. Since most of these virulence-associated substitutions were absent in the earliest pdmH1N1s, it has been speculated that the virus could acquire some of these mutations, potentially resulting in the emergence of more pathogenic viruses. Such virulence markers could be acquired by gene reassortment with cocirculating influenza A viruses, or by mutation. The influenza virus polymerase genes, in particular PB2, have been shown to be important determinants of the virulence of the highly pathogenic avian influenza (HPAI) H5N1 and H7N7 viruses and the transmission of the 1918 H1N1 Spanish influenza virus (17, 26, 34, 51). One of the most commonly identified virulence markers to date is E627K in PB2. The glutamic acid (E) residue is generally found in avian influenza viruses, while human viruses have a lysine (K), and this mutation was described as a determinant of host range in vitro (48). Given that all human and many zoonotic influenza viruses of the last century contained 627K, it was surprising that the pdmH1N1 had 627E. In addition, an aspartate (D)-to-asparagine (N) substitution at position 701 (D701N) of PB2 has previously been shown to expand the host range of avian H5N1 virus to mice and humans and to increase virus transmission in guinea pigs (26, 46). Like E627K, D701N was absent in the genome of pdmH1N1. Thus, the pdmH1N1 was the first known human pandemic virus with 627E and 701D, and it has been speculated that pdmH1N1 could mutate into a more virulent form by acquiring one of these mutations or both. Recently, it was shown that neither E627K nor D701N in PB2 of pdmH1N1 increased its virulence in ferrets and mice (18). The PB1-F2 protein has previously also been associated with high pathogenicity of the 1918 H1N1 and HPAI H5N1 viruses (8). The PB1-F2 protein of the pdmH1N1 is truncated due to premature stop codons. However, restoration of the PB1-F2 reading frame did not result in viruses with increased virulence (15). The NS1 protein of pdmH1N1 is also truncated due to a stop codon and, as a result, does not contain a PDZ ligand domain that is involved in cell-signaling pathways and has been implicated in the pathogenicity of 1918 H1N1 and HPAI H5N1 viruses (5, 8, 21). Surprisingly, restoration of a full-length version of the NS1 gene did not result in increased virulence in animal models (16). Mutations affecting virulence and host range have further frequently been mapped to hemagglutinin (HA) and neuraminidase (NA) in relation to their interaction with α2,3- or α2,6-linked sialic acids (SAs), the virus receptors on host cells (17, 32, 35, 50). The HA gene of previous pandemic viruses incorporated substitutions that allow efficient attachment to α2,6-SAs—the virus receptor on human cells—compared to ancestral avian viruses that attach more efficiently to α2,3-SAs (35, 47, 50).To search for mutations of potential importance to public health, numerous laboratories performed genome sequencing of pdmH1N1s, resulting in the real-time accumulation of information on emergence of potential virulence markers. Of specific interest were reports on amino acid substitutions from aspartic acid (D) to glycine (G) at position 222 (position 225 in H3) in HA of pdmH1N1. This substitution was observed in a fatal case of pdmH1N1 infection in June 2009 in the Netherlands (M. Jonges et al., unpublished data). Between July and December 2009, viruses from 11 (18%) of 61 cases with severe disease outcome in Norway have also been reported to harbor the D222G substitution upon direct sequencing of HA in clinical specimens. Such mutant viruses were not observed in any of 205 mild cases investigated, and the frequency of detection of this mutation was significantly higher in severe cases than in mild cases (23). In Hong Kong, the D222G substitution was detected in 12.5% (6) and 4.1% (31) of patients with severe disease and in 0% of patients with mild disease, in two different studies without prior propagation in embryonated chicken eggs. In addition to Norway and Hong Kong, the mutation has been detected in Brazil, Japan, Mexico, Ukraine, and the United States (56). Thus, D222G in HA could be the first identified “virulence marker” of pdmH1N1. pdmH1N1 with D222G in HA have not become widespread in the population, although they were detected in several countries. However, D222G in HA is of special interest, since it has also been described as the single change in HA between two strains of the “Spanish” 1918 H1N1 virus that differed in receptor specificity (47). Furthermore, upon propagation in embryonated chicken eggs, pdmH1N1 can acquire the mutation rapidly, presumably because it results in virus adaptation to avian (α2,3-SAs) receptors (49). The presence of the substitution in pdmH1N1s in the human population and its potential association with more severe disease prompted us to test its effect on pdmH1N1 receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models.     

Quantitative Characterization of Glycan-Receptor Binding of H9N2 Influenza A Virus Hemagglutinin

Avian influenza subtypes such as H5, H7 and H9 are yet to adapt to the human host so as to establish airborne transmission between humans. However, lab-generated reassorted viruses possessing hemagglutinin (HA) and neuraminidase (NA) genes from an avian H9 isolate and other genes from a human-adapted (H3 or H1) subtype acquired two amino acid changes in HA and a single amino acid change in NA that confer respiratory droplet transmission in ferrets. We previously demonstrated for human-adapted H1, H2 and H3 subtypes that quantitative binding affinity of their HA to α2→6 sialylated glycan receptors correlates with respiratory droplet transmissibility of the virus in ferrets. Such a relationship remains to be established for H9 HA. In this study, we performed a quantitative biochemical characterization of glycan receptor binding properties of wild-type and mutant forms of representative H9 HAs that were previously used in context of reassorted viruses in ferret transmission studies. We demonstrate here that distinct molecular interactions in the glycan receptor-binding site of different H9 HAs affect the glycan-binding specificity and affinity. Further we show that α2→6 glycan receptor-binding affinity of a mutant H9 HA carrying Thr-189→Ala amino acid change correlates with the respiratory droplet transmission in ferrets conferred by this change. Our findings contribute to a framework for monitoring the evolution of H9 HA by understanding effects of molecular changes in HA on glycan receptor-binding properties.     

Computational Identification of Antigenicity-Associated Sites in the Hemagglutinin Protein of A/H1N1 Seasonal Influenza Virus

The antigenic variability of influenza viruses has always made influenza vaccine development challenging. The punctuated nature of antigenic drift of influenza virus suggests that a relatively small number of genetic changes or combinations of genetic changes may drive changes in antigenic phenotype. The present study aimed to identify antigenicity-associated sites in the hemagglutinin protein of A/H1N1 seasonal influenza virus using computational approaches. Random Forest Regression (RFR) and Support Vector Regression based on Recursive Feature Elimination (SVR-RFE) were applied to H1N1 seasonal influenza viruses and used to analyze the associations between amino acid changes in the HA1 polypeptide and antigenic variation based on hemagglutination-inhibition (HI) assay data. Twenty-three and twenty antigenicity-associated sites were identified by RFR and SVR-RFE, respectively, by considering the joint effects of amino acid residues on antigenic drift. Our proposed approaches were further validated with the H3N2 dataset. The prediction models developed in this study can quantitatively predict antigenic differences with high prediction accuracy based only on HA1 sequences. Application of the study results can increase understanding of H1N1 seasonal influenza virus antigenic evolution and accelerate the selection of vaccine strains.     

Amino Acid Substitutions That Affect Receptor Binding and Stability of the Hemagglutinin of Influenza A/H7N9 Virus

Receptor-binding preference and stability of hemagglutinin have been implicated as crucial determinants of airborne transmission of influenza viruses. Here, amino acid substitutions previously identified to affect these traits were tested in the context of an A/H7N9 virus. Some combinations of substitutions, most notably G219S and K58I, resulted in relatively high affinity for α2,6-linked sialic acid receptor and acid and temperature stability. Thus, the hemagglutinin of the A/H7N9 virus may adopt traits associated with airborne transmission.     

Evolution of the Hemagglutinin Protein of the New Pandemic H1N1 Influenza Virus: Maintaining Optimal Receptor Binding by Compensatory Substitutions

Pandemic influenza A H1N1 (pH1N1) virus emerged in 2009. In the subsequent 4 years, it acquired several genetic changes in its hemagglutinin (HA). Mutations may be expected while virus is adapting to the human host or upon evasion from adaptive immune responses. However, pH1N1 has not displayed any major antigenic changes so far. We examined the effect of the amino acid substitutions found to be most frequently occurring in the pH1N1 HA protein before 1 April 2012 on the receptor-binding properties of the virus by using recombinant soluble HA trimers. Two changes (S186P and S188T) were shown to increase the receptor-binding avidity of HA, whereas two others (A137T and A200T) decreased binding avidity. Construction of an HA protein tree revealed the worldwide emergence of several HA variants during the past few influenza seasons. Strikingly, two major variants harbor combinations of substitutions (S186P/A137T and S188T/A200T, respectively) with opposite individual effects on binding. Stepwise reconstruction of the HA proteins of these variants demonstrated that the mutations that increase receptor-binding avidity are compensated for by the acquisition of subsequent mutations. The combination of these substitutions restored the receptor-binding properties (avidity and specificity) of these HA variants to those of the parental virus. The results strongly suggest that the HA of pH1N1 was already optimally adapted to the human host upon its emergence in April 2009. Moreover, these results are in agreement with a recent model for antigenic drift, in which influenza A virus mutants with high and low receptor-binding avidity alternate.