Diversifying selective pressure on influenza B virus hemagglutinin

Influenza B virus hemagglutinin (HA) is a major surface glycoprotein with frequent amino acid substitutions. However, the roles of antibody selection in the amino acid substitutions of HA were still poorly understood. In order to gain insights into this important issue, an analysis was conducted on a total of 271 HA₁ sequences of influenza B virus strains isolated during 1940–2007. In this analysis, phylogenetic analysis by maximum likelihood (PAML) package was used to detect the existence of positive selection and to identify positively selected sites on HA₁. Strikingly, all the positively selected sites were located in the four major epitopes (120‐loop, 150‐loop, 160‐loop, and 190‐helix) of HA identified in previous studies, thus supporting a predominant role of antibody selection in HA evolution. Of particular significance is the involvement of the 120‐loop in positive selection, which may become increasingly important in future field isolates. Despite the absence of different subtypes, influenza B virus HA continued to evolve into new sublineages, within which the four major epitopes were targeted selectively in positive selection. Thus, any newly emerging strains need to be placed in the context of their evolutionary history in order to understand and predict their epidemic potential. J. Med. Virol. 81:114–124, 2009. © 2008 Wiley‐Liss, Inc.     

Antigenic determinants of the hemagglutinin of influenza virus type B

A comparative immunological analysis of 3 influenza type B virus strains, B/Lee/40, B/M/8/52, and B/Victoria/70 was made. The presence of at least 3 antigenic determinants in hemagglutinins of the viruses under study was established by the method of selective adsorption. One of the determinants was type-specific and was shared by all the three strains, the other two determined the group- and strain-specificity of the viruses studied.     

Antigenic determinants in influenza virus hemagglutinin.

Three antigenic determinants were revealed in H3 hemagglutinin of influenza A viruses isolated from 1968 to 1975. One of them was common for all viruses, and two others specified differences between the viruses possessing H3 hemagglutinin.     

Antigenic relationships between strains of influenza B virus.

An immunological relationship between strains of influenza B virus, considerably differing from one another in haemagglutination inhibition (HI) and virus neutralization (VN) tests, was established. The relationships were also evaluated based on the ability of influenza B viruses to replicate in the lungs of mice immunized with strains possessing antigenically distinct haemagglutinin. There was no substantial difference in the protection of animals immunized with homologous or heterologous strains. Studies on the character of the immunological response of men convalescent after influenza B infection or after vaccination showed an antibody increase to both the epidemic virus and chronologically remote viruses considerably differing in antigenic properties. The data obtained suggest that influenza B viruses isolated from 1940 to 1975 belong to one antigenic subtype.     

Immunogenicity of the antigenic determinants of influenza virus type B hemagglutinin

The immunogenic activity of antigenic determinants of influenza type B viruses in primary and booster immunizations was studied. In primary immunization, the highest immunogenicity was found with the type- and strain-specific antigenic determinants whereas the antigenic determinants of group specificity were less immunogenic. After booster immunizations the animal sera had antibody to all 3 antigenic determinants.     

Preparation of influenza B virus recombinant strains

The study of antigenic and biologic properties of influenza B epidemic viruses isolated in 1979 and 1983 and laboratory strain B/Lee/40 has revealed some differences in their biologic properties. The most marked changes have been found in the haemagglutinin (HA) and neuraminidase (NA) indicating that influenza B viruses underwent dramatic antigenic drifts during the period in question. The strains obtained by genetic recombination have inherited surface antigens of epidemic influenza B/Singapore/222/79 and B/USSR/100/83 viruses and preserved the HA thermolability inherent to these viruses. They have, however, acquired the marker of reproduction in chick embryos and the immunogenicity from the donor strain B/Lee/40. These recombinants can be, therefore, recommended as vaccine strain candidates.     

Separation of two polypeptide chains from the hemagglutinin subunit of influenza virus

Particles of the BEL(Ao) strain of influenza virus (grown in the allantoic sac of embryonated chicken eggs) were disrupted with cold sodium dodecyl sulphate (SDS), and the hemagglutinin subunits were isolated by electrophoresis on cellulose acetate strips. The hemagglutinin subunits were found to contain two different polypeptide chains with molecular weights of approximately 60,000 and 21,000. In the intact subunits the heavy chain was joined by disulphide bond(s) to the light chain to form a dimer, and each hemagglutinin subunit contained two of these dimers. The two chains were separated by SDS-polyacrylamide gel electrophoresis in the presence of dithiothreitol or, on a preparative scale, by centrifugation on a guanidine hydrochloridedithiothreitol density gradient. The two chains were similar in amino acid composition, with the exception that the heavy chain contained about 9 times more proline than the light chain. The heavy chain also contained much more glucosamine. Maps of the tryptic peptides from the two chains were quite different, indicating that they differed in amino acid sequence.     

Genetic variation of the hemagglutinin of avian influenza virus H9N2

Avian influenza virus H9N2 has become the dominant subtype of influenza which is endemic in poultry. The hemagglutinin, one of eight protein-coding genes, plays an important role during the early stage of infection. The adaptive evolution and the positively selected sites of the HA (the glycoprotein molecule) of H9N2 subtype viruses were investigated. Investigating 68 hemagglutinin H9N2 avian influenza virus isolates in China and phylogenetic analysis, it was necessary that these isolates were distributed geographically from 1994, and were all derived from the Eurasian lineage. H9N2 avian influenza virus isolates from domestic poultry in China were distinct phylogenetically from those isolated in Hong Kong, including viruses which had infected humans. Seven amino acid substitutions (2T, 3T, 14T, 165D, 197A, 233Q, 380R) were identified in the HA possibly due to positive selection pressure. Apart from the 380R site, the other positively selected sites detected were all located near the receptor-binding site of the HA1 strain. Based on epidemiological and phylogenetics analysis, the H9N2 epidemic in China was divided into three groups: the 1994-1997 group, the 1998-1999 group, and the 2000-2007 group. By investigating these three groups using the maximum likelihood estimation method, there were more positive selective sites in the 1994-1997 and 1998-1999 epidemic group than the 2000-2007 groups. This indicates that those detected selected sites are changed during different epidemic periods and the evolution of H9N2 is currently slow. The antigenic determinant or other key functional amino acid sites should be of concern because their adjacent sites have been under positive selection pressure. The results provide further evidence that the pathogenic changes in the H9N2 subtype are due mainly to re-assortment with other highly pathogenic avian influenza viruses.     

Enumeration of antigenic sites of influenza virus hemagglutinin.

The antigenic sites on the hemagglutinin of X-31 (H3) influenza virus have been defined by using a competitive radioimmunoassay with a panel of monoclonal antibodies which includes those known to select variants with substitutions of particular amino acids. The capacity of each monoclonal antibody to block the binding of other radioiodinated monoclones to purified hemagglutinin permitted classification of the panel into four separate groups, each of which defined a particular antigenic site on the hemagglutinin molecule. Three of these are located on the polypeptide backbone and correspond to the "hinge," the "loop," and the "tip/interface" of the X-ray crystallographic model of Wiley et al. (Nature [London] 289:373-378, 1981). Nonreciprocal blocking of certain anti-interface antibodies by anti-loop antibody suggests that much of the exposed surface of the head of the hemagglutinin molecule extending from the loop to the interface may be a continuum of epitopes. A fourth antigenic site is carbohydrate in nature, presumably situated on the antigenic oligosaccharide side chains. These four domains are in addition to two antigenic sites defined by monoclonal antibodies that inhibit neither hemagglutination nor infectivity (Breschkin et al., Virology 113:130-140, 1981;' Yewdell et al., Nature [London] 279:246-248, 1979).     

Antigenic and molecular characterization of subtype H13 hemagglutinin of influenza virus

Influenza A viruses with subtype H13 hemagglutinin display an unusual host range. Although common in shorebirds, they are very rare or absent in wild ducks; additionally, H13 viruses have been isolated from a whale. To study the molecular basis for this host range, we have determined the complete nucleotide sequences of the hemagglutinin genes of three H13 influenza viruses from different species or geographical areas: A/gull/Maryland/77, A/gull/Astrachan (USSR)/84, and A/pilot whale/Maine/84. Based on the deduced amino acid sequences, H13 hemagglutinin shares the basic structure of other type A hemagglutinin subtypes such as H3, but has clearly diverged from other completely sequenced subtypes. Unique features of H13 hemagglutinin include the occurrence, near the receptor binding pocket, of residues Arg/Lys-227 and Trp-229 (H3 numbering); the significance of these are unknown. The sequence of the HA1-HA2 cleavage site resembles those of avirulent avian influenza viruses. The whale H13 hemagglutinin is similar to those from gulls, supporting the hypothesis that influenza viruses from avian sources can enter marine mammal populations but are probably not permanently maintained there. Antigenic analysis using a panel of monoclonal antibodies suggests that, like other subtypes, H13 viruses are heterogeneous, with different antigenic variants predominating in the eastern versus the western hemispheres.     

Phylogenetic analysis of influenza B virus in Taiwan, 1997 to 2001.

Seventeen strains of influenza B virus were isolated and identified from 1997 to 2001. Throat swabs were collected in children who presented in medical centers in both central and northern parts of Taiwan. To clarify the molecular characteristics of these isolates, both partial hemagglutinin (HA) gene and nonstructural (NS) gene nucleotide sequences were cloned and subjected to nucleotide sequence analysis. The phylogenetic analysis of the HA gene revealed that 16 out of 17 strains were similar to B/Yamagata/16/88-like virus, but grouped together to form an independent cluster. Only one strain, B/Taiwan/21706/97, was similar to the B/Victoria/2/87-like lineage. In addition, all isolates, except for B/Taiwan/21706/97, were similar to B/Beijing/184/93 and B/Yamanashi/166/98, which were chosen as the recommended vaccine strains in 1999 and 2001. In contrast, the NS gene of these isolates was evolved from B/Guangdong/8/93. Based on the accumulation of antigenic drift in our isolates, we conclude that influenza B virus is still prevalent in Taiwan and the accumulation of nucleotide mutations indicated that our isolates form a new cluster that evolved from the YA88 lineage.     

Alterations in the hemagglutinin associated with adaptation of influenza B virus to growth in eggs

In 1943 Burnet reported on changes in the hemagglutinating properties of human influenza virus which occurred during adaptation of the virus to growth in chicken eggs. Only recently has direct evidence been presented that these changes affect the antigenic properties of the virus. Schild et al. (G. C. Schild, J. S. Oxford, J. C. deJong, and R. G. Webster (1983), Nature (London) 303, 706-709) demonstrated that egg adaptation of influenza B virus selects variants which are antigenically distinct from virus grown from the same source in mammalian cells. The molecular changes in the hemagglutinin (HA) of influenza B virus associated with adaptation to growth in eggs have now been identified. A specific glycosylation site at the distal tip of the HA of influenza B virus grown exclusively in mammalian cell culture is lost or altered during egg adaptation. Since the HA functions in adsorption of virus to cells, it is concluded that removal or modification of an oligosaccharide structure at this position is required for influenza B virus to attach to and infect the allantois cells of the egg and that this has important implications for the antigenic configuration of the molecule.     

The polypeptides of influenza virus. VII. Synthesis of the hemagglutinin

Post-translational cleavage of the influenza viral glycoprotein HA occurs to different extents in different systems, varying not only for a particular virus strain grown in different host cells, but also for two strains of virus grown in the same host cell. Preparations of virus in which the HA is not substantially cleaved contain hemagglutinating and infectious virions. Cleavage occurs at different sites in the HA molecule of different virus strains.The hemagglutinin glycoprotein HA is always found in association with cytoplasmic membranes and becomes rapidly incorporated into plasma membranes. Following a 10-min pulse-label, there is already about half as much HA in preparations of plasma membranes as eventually accumulates there during a 90-min chase. Membrane preparations which appear to be mainly composed of smooth endoplasmic reticulum are greatly enriched for HA while plasma membranes contain HA and the other major viral proteins. At 24 hr after infection, the amount of HA or its cleavage products in BHK21 cells infected with Bel or WSN represents a much smaller proportion of the total viral protein than the proportion of HA in purified virions. The same is true for the membrane protein, M, whereas NP is present in excess in the infected cell.Inhibition of protein synthesis by puromycin stops the incorporation of glucosamine into Bel-infected HeLa cells almost immediately, suggesting that glycosylation of HA occurs quickly. However, fucose continues to be incorporated for apporoximately 10–15 min after protein synthesis has been blocked by puromycin or after glycosiliation has been inhibited by glucosamine hydrochloride.     

Preparation and characterization of rabies virus hemagglutinin.

Rabies virus glycoprotein, released by treatment with Triton X-100, was isoelectrically focused in a sucrose gradient containing the nonionic detergent octylglucoside. Removal of the detergent by dialysis resulted in aggregates of variable size and shape. The hemagglutinating activity of this preparation was approximately sixfold higher than that of the intact virus. The protein with hemagglutinating activity and with a buoyant density of 1.237 consisted solely of polypeptide chains of the G-protein and contained 0.38% phospholipids and 16 ng of Triton X-100 per mg of protein. In the National Institutes of Health test the hemagglutinin conferred a significantly higher protective activity than detergent-associated glycoprotein and was as effective as an inactivated virus vaccine. However, after the application of a single dose of hemagglutinin, the onset of protection was delayed by approximately 7 days when compared with inactivated virus vaccine.     

Phenotyte of epidemic influenza B virus strains isolated in different years

The reproducing ability at elevated temperatures (non-ts phenotype) was examined for 38 influenza B virus strains isolated in different years in different countries. Out of the 7 strains isolated in 1940 to 1973, only one showed temperature-sensitivity of reproduction (a ts phenotype). In 1984 to 1988, the proportion of temperature-sensitive strains increased up to 55% (6 of 11). Since the late 1990s, the majority (90%) of the study influenza B viruses demonstrated a pronounced ts phenotype. Influenza B virus strains were also examined for their resistance to serum inhibitors. Prior to the divergence of influenza B viruses into two lines: B/Jamagata and B/Victoria, the epidemic viruses exhibited a high resistance to nonspecific inhibitors of normal equine serum. This property was also preserved in all study B/Victoria strains; however, 83% of the B/Jamagata viruses were inhibitor-sensitive. The present study has demonstrated the heterogenicity of epidemic influenza B viruses in temperature- and inhibitor-sensitivity.     

Structural polypeptides of antigenically distinct strains of influenza B virus

Analyses of the polypeptide composition of influenza B viruses by 13 per cent SDS-polyacrylamide gel electrophoresis are reported. The viruses contained polypeptides of eight species ranging in molecular weight from 27,000 to 78,000. Four of them were glocypeptides and were selectively removed from the surface of the virion by Bromelain treatment. One of the blycopeptides was identified as viral neuraminidase. Three antigenically distinct strains of influenza virus, B/Lee/40, B/Massachusetts/1/71 and B/Hong Kong/5/72, showed an essentially identical electrophoretic picture, although strain-to-strain difference was observed in the migration rate of HA1 and HA2 polypeptides.     

Genetic variation among human strains of influenza C virus isolated in Japan

The RNA genomes of sixteen human strains of influenza C virus isolated in Japan between 1964 and 1983 were compared by SDS-polyacrylamide gel electrophoresis and oligonucleotide fingerprinting. A high degree of genetic variation was observed among the strains analysed. However, there were some strains with the genomes closely related to one another, and they could be divided into two groups. The first group consists of C/Shizuoka/79, C/Kanagawa/1/81 and four strains of C/Yamagata/81. The 1981 strains of this group were all isolated in March of the year. The second one consists of C/Kyoto/41/82, C/Nara/82 and C/Hyogo/1/83 that were isolated between February 1982 and December 1983. Little or no difference was observed in the genomes of the same group, while the difference was evident between two groups. The Aichi/1/81 strain isolated in November 1981 had a genome distantly related to either of these two groups. Thus three different types of influenza C virus were isolated during the period of 12 mth from March 1981 to February 1982, suggesting that multiple influenza C viruses with distant genetic relationship were circulating at the same time in Japan.     

抗禽流感病毒H7亚型血凝素特异性单克隆抗体的研制

目的:研制禽流感病毒H7亚型血凝素特异性单克隆抗体(mAb)。方法:以H7亚型禽流感诊断抗原为免疫原免疫6～8周雌性BALB/c小鼠,末次加强免疫后取其脾细胞与骨髓瘤细胞Sp2/0-Ag-14进行融合。通过HA和HI试验筛选阳性克隆。应用HI试验和Western blot试验测定mAb的反应性和特异性。结果:共获得4株分泌抗AIVH7亚型HAmAbs的杂交瘤细胞株,分别命名为2E2、2A4、5F5、7G5。这些mAb的腹水HI效价在5×27～5×211之间,其中2E2属于IgM亚类,2A4属于IgG1亚类,5F5、7G5属于IgG2a亚类。Western blot分析结果显示,4株AIVH7亚型HAmAb能与AIVH7蛋白在Mr75000处反应,但不与新城疫病毒(NDV)蛋白发生反应,表明这些mAb能特异性识别AIVH7亚型HA。mAbHI反应性测定结果表明:4株mAb中,2E2、5F5、7G5只与H7亚型AIV发生特异性HI反应,而不与其他亚型AIV以及NDV、传染性支气管炎病毒(IBV)反应,显示出良好的特异性;而2A4除了与H7亚型AIV反应外,还与H15N8标准株发生低水平交叉反应。结论:这些mAb不仅为H7亚型AIV的HA结构分析提供了工具,而且为建立快速廉价的H7亚型禽流感诊断方法提供了核心试剂。