Noncumulative sequence changes in the hemagglutinin genes of influenza C virus isolates

Sequence analysis and comparison of hemagglutinin (HA) genes of different influenza C viruses isolated between 1947 and 1983 reveals that (1) the extent of difference among the HA genes is independent of the year in which these viruses were isolated and that (2) changes in the HA genes do not appear to accumulate with time. These results suggest that epidemiologically dominant variants of influenza C viruses do not emerge successively with time and that C virus variants derived from multiple evolutionary pathways cocirculate at any one time. Thus the epidemiology of influenza C viruses differs markedly from that of influenza A viruses, which is characterized by the emergence of successive variants. Based on the nucleotide sequence data, we propose different evolutionary models for influenza A and influenza C viruses.     

Codon usage bias and recombination events for neuraminidase and hemagglutinin genes in Chinese isolates of influenza A virus subtype H9N2

The representative complete neuraminidase sequences (NA) of 138 Chinese isolates and hemagglutinin sequences (HA) of 128 Chinese isolates of influenza A virus subtype H9N2 were analyzed to reveal codon usage bias and recombination events. A plot of the effective number of codons (ENC) against G + C context at the third position of the codons (ENC-plot) and correlation analysis demonstrated that mutational pressure is the main factor determining the codon usage bias in the H9N2 subtype. The high ENC values (51.31 for NA and 48.83 for HA) indicated that the codon usage bias in Chinese H9N2 isolates was very low. Plots of correspondence analysis and an ENC plot indicated that the HA and NA genes had different codon usage bias. Recombination analysis revealed two possible recombination events for HA and NA gene sequences in the Chinese isolates. Subsequent phylogenetic analysis further supported the validity of these recombination events. In addition, one false-positive recombinant for HA gene was found, which may have been derived from contamination during PCR amplification.     

Distinct hemagglutinin and neuraminidase epitopes involved in antigenic variation of recent human parainfluenza virus type 2 isolates.

A panel of fourteen neutralizing anti-HN monoclonal antibodies (mAbs) to the prototype Greer strain of human parainfluenza virus type 2 (PI2) was used to determine the extent of antigenic variation in recent virus isolates. Competitive binding analysis with the mAbs indicated the presence of at least five distinct antigenic sites (I to V) on the HN glycoprotein molecule. MAbs recognizing different antigenic sites were found to be associated with the hemagglutinin (sites I, IV and V), hemagglutinin and neuraminidase (site II), or neuraminidase (site III) activities. The location of two distinct epitopes identifying the neuraminidase sites (II and III) was further verified from the generation of escape mutants. Antibodies directed to sites I and III failed to show any detectable binding or neutralizing activity against a number of natural PI2 virus isolates collected in Texas between 1986 and 1987. Interestingly, these natural variants, unlike the prototype virus, did not show any detectable neuraminidase activity with fetuin as a substrate and the enzyme activity was only detected with N-acetylneuramin-lactose as an alternative substrate. Despite the observed variation in the antigenic sites, primary infection with the prototype virus or the natural variants generated a protective immune response against challenge infection with the other virus strains.     

Lymphocyte responses to hemagglutinin and neuraminidase subunits of influenza virus.

The responses of peripheral lymphocytes to purified hemagglutinin and neuraminidase subunits and other components of an influenza. A virus were measured in 21 normal adults and compared with antibody titers. All had influenza antibodies and demonstrated influenza antigen recognition by lympho-proliferative responses. There was a significantly greater response to the two purified influenza virus surface antigens, hemagglutinin and neuraminidase, than to either whole intact virus or its separated subviral core. No correlation between magnitudes of antibody titers and lymphocyte responsiveness was observed.     

Codon volatility of hemagglutinin genes of H5N1 avian influenza viruses from different clades

Codon volatility is a method recently developed to estimate selective pressures on proteins on the basis of their synonymous codon usage. Volatility of a codon was defined as the fraction of single nucleotide substitutions that would be nonsynonymous. Higher volatility may indicate that the gene has been under more positive selection in the recent past. We analyzed volatility of hemagglutinin genes of H5N1 viruses in the recent outbreaks and observed differences in the volatility among viruses of different clades. The codon volatility of subclade 2.1 viruses from Indonesia was the lowest among all H5N1 clades and subclades. Time series analyses since the beginning of the epidemic in 2004 showed that codon volatility of subclade 2.1 has gradually decreased, while those of other major clades have been increasing. This may reflect differences in the recent evolution of these viruses.     

Mismatched hemagglutinin and neuraminidase specificities in recent human H3N2 influenza viruses

The hemagglutinin (HA) of influenza viruses initiates infection by binding to sialic acid on the cell surface via alpha2,6 (human) or alpha2,3 (avian) linkage. The influenza neuraminidase (NA) can cleave both alpha2,3- and alpha2,6-linked sialic acids, but all influenza NAs have a marked preference for the non-human alpha2,3 linkage. Recent H3N2 influenza viruses have lost the ability to agglutinate chicken red blood cells. To determine if changes in HA specificity or affinity correlate with NA specificity or activity, we examined red cell binding and elution of a series of H3N2 viruses. We found that the NA activity of many influenza viruses does not release binding by their HA. In some egg-adapted strains, lack of elution correlates with low levels of viral NA activity, and these elute rapidly when bacterial NA is added. However, a Fujian-like virus, A/Oklahoma/323/03, does not elute by its own NA or with Vibrio cholerae sialidase, and it binds to red cells pre-treated with V. cholerae sialidase. It elutes after addition of the broad specificity Micromonospora viridifaciens sialidase. Human glycophorin inhibits A/Oklahoma/323/03 hemagglutination 6-fold better than fetuin. We conclude that specific forms of sialic acid are used as receptor by recent human H3N2 influenza viruses, perhaps involving branched alpha2,6 sialic acid or alpha2,8 sialic acid structures on O-linked carbohydrates. The virus itself has no O-linked glycans, so even though the NA is not able to cleave receptors on cells, the viruses will not self-aggregate. It will be important to monitor efficacy of neuraminidase inhibitors in case there are NA-resistant receptors in the human respiratory tract that allow the viruses to be less dependent on NA activity.     

Identification of amino acids in highly pathogenic avian influenza H5N1 virus hemagglutinin that determine avian influenza species specificity

To test the role of neutralizing antibodies (nAbs) and receptor adaptation in interspecies transmission of influenza virus, two H5N1 strains, isolated from human and avian hosts, with four amino acid differences in hemagglutinin (HA) and seven HA mutations were studied. We found that a mutation at amino acid position 90 in the H5N1 HA, outside the receptor-binding domain (RBD), could simultaneously induce changes in the RBD conformation to escape from nAb binding and alter the receptor preference through long-range regulation. This mutation was deemed a “key event” for interspecies transmission. It is likely a result of positive selection caused by antibodies, allowing the original invasion by new species-specific variants. A mutation at amino acid position 160 in the RBD only induced a change in receptor preference. This mutation was deemed a “maintaining adaptation”, which ensured that influenza virus variants would be able to infect new organisms of a different species successfully. The mutation is the result of adaptation caused by the receptor. Our results suggest that continuing occurrence of these two types of mutations made the variants persist in the new host species.     

Disulfide maturation of the glycoproteins of influenza virus hemagglutinin and neuraminidase in infected cells

Fractionation by polyacrylamide gel electrophoresis (PAGE) demonstrated that in the infected cells the newly synthesized influenza virus glycoproteins, hemagglutinin (HA) and neuraminidase (NA), differ from mature proteins of virus particles. After some time of life in the cells the differences are levelled. Since this phenomenon was demonstrable only in an analysis under the conditions favourable for the retention of disulphide bonds, it was designated as "disulphide maturation" of glycoproteins. Two causes of disulphide maturation of HA are considered: posttranslational folding of molecules conducive to drawing closer of the oxidizable thiol groups, and gradual loss of sensitivity to endogenous reducing agents. As for NA, the observed maturation here is the result of disulphide dimerization of monomers. Some factors affecting disulphide maturation of glycoproteins have been studied.     

Divergent evolution of hemagglutinin and neuraminidase genes in recent influenza A:H3N2 viruses isolated in Canada

Limited information is available concerning the molecular drift of the influenza neuraminidase (NA) genes. We report on the genetic variability of the NA gene from 31 influenza A:H3N2 viruses isolated in the Province of Québec (Canada) during the last three flu seasons (1997-2000). Amino acid substitutions within the NA protein were observed at rates of 1.01% and 0.45% between strains of the 1997-1998 and 1998-1999 seasons and between those of the 1998-1999 and 1999-2000 seasons, respectively. In most strains (28/31), amino acid changes occurred within at least one of four codons (197, 339, 370, and 401) previously implicated as antigenic sites. The 8 functional and 10 framework residues that compose the catalytic site of the NA enzyme were completely conserved over the study period. All isolates contained the seven conserved asparagine-linked glycosylation sites found in the NA of the progenitor A/Hong Kong/8/68 strain. In addition, most strains (30/31) had an eighth potential glycosylation site at position 329, whereas a ninth one was found at position 93 in 16 strains. The NA of all strains in this study was related to that of the vaccine strain A/Beijing/353/89, whereas the HA genes of these strains were related to the A/Beijing/32/92 vaccine strain. Thus, it appears that recent influenza strains continue to evolve from a reassortant produced during the cocirculation of the two above variants. Interestingly, some strains whose HA genes were closely related showed significant differences in their NA genes and conversely. This study confirms the importance of analyzing both the NA and the HA genes to assess the evolution of recent influenza epidemic strains.     

Strain-specific differences in the effect of influenza A virus neuraminidase on vector-expressed hemagglutinin

Summary.  In order to evaluate the efficiency of the removal of sialic acid residues from the influenza virus hemagglutinin by the viral neuraminidase in the course of the virus replication cycle, CV-1 cells expressing the hemagglutinin of H7 subtype from an SV40-based vector were superinfected with influenza virus strain A/Duck/Ukraine/63 (H3N8) or A/USSR/90/77 (H1N1). Vector-expressed hemagglutinin was immunoprecipitated from cell lysates and analyzed by polyacrylamide gel electrophoresis. The data indicate that the removal of sialic acid residues from the vector-expressed H7 hemagglutinin by N1 neuraminidase of A/USSR/90/77 virus in the course of the virus replication cycle is incomplete. The results are discussed in connection with previously published data showing that the low activity of NA in wild-type influenza virus results in incomplete removal of sialic acid residues from virion components. Received July 8, 1998 Accepted November 26, 1998     

Preparation and immunogenicity of an influenza virus hemagglutinin and neuraminidase subunit vaccine.

Vaccines containing hemagglutinin and neuraminidase subunits were prepared from the A/Port Chalmers/1/73 (H3N2) strain of influenza virus. The virus particles were disrupted with ammonium deoxycholate and the matrix protein, which was insoluble in this detergent, was removed by centrifugation. Following removal of deoxycholate, the hemagglutinin and neuraminidase subunits aggregated by their hydrophobic ends, forming mixed clusters. These were then freed from nucleocapsids by electrophoresis. The separated nucleocapsid protein and the matrix protein were thus obtained as byproducts in a highly purified form suitable for the production of antisera and for structural studies. The hemagglutinin and neuraminidase subunits were as effective as intact inactivated virus (at equivalent concentration) in eliciting a late primary antibody response when injected in saline into rabbits. In hamsters, the subunits failed to induce antibody when injected in saline (in contrast to intact virus), but the immune response to the subunits could be potentiated by the simultaneous injection of an intact heterologous influenza A or B virus.     

Molecular cloning and analysis of the N5 neuraminidase subtype from an avian influenza virus

The neuraminidase (NA) gene from the prototype N5 influenza virus, A/Shearwater/Australia/72, has been cloned and completely sequenced. An open reading frame of 1404 bp (468 amino acids) is flanked by 20-bp 5'- and 31-bp 3'-untranslated regions. The deduced amino acid sequence of the N5 gene was compared with sequences from N2, N1, N7, N8, and N9 subtypes. One hundred thirteen amino acid residues (24%) are completely conserved across subtypes and include active site residues, cysteines, potential glycosylation sites, and certain glycines which suggests that these subtypes share a common ancestor and adopt the same 3-D conformation. Three groups can be assigned from amino acid homologies: (i) N5, N8, N1; (ii) N7, N9; and (iii) N2 where the percentage identity within groups is 55-68% and between groups is 40-46%, the N5-N8 pair bearing the closest identity (68%). Phylogenetic analysis suggests that these groups diverged concurrently.     

Characterization of temperature sensitive influenza virus mutants defective in neuraminidase

Two temperature sensitive mutants, ts3 and ts11, of the WSN (HON1) strain of influenza virus, belonging to the same recombination group, have a 1000- to 10,000-fold lower infectivity titer and lack hemagglutinating and neuraminidase activity when grown at nonpermissive temperature (39.5°), compared with virus grown at permissive temperature (33°). The patterns of viral polypeptides synthesized in cells infected with these mutants are similar to those found with wild type virus. Neuraminidase activity of the mutant viruses is more temperature labile than the enzyme of the wild type. Despite the low yield, morphologically intact virus particles are found at 39.5°, but in contrast to virus grown at 33° large aggregates of virus accumulate near the cell surface. These aggregated virus particles contain neuraminic acid as demonstrated by a colloidal iron stain. Thus, it is likely that a neuraminic acid containing protein serves as receptor for the hemagglutinin of other virus particles, resulting in the extensive aggregation. Hemagglutinating activity of virus grown at 39.5° is restored by treatment of the aggregates with neuraminidase from Vibrio cholerae. These results suggest that the lack of hemagglutinating activity of mutant virus grown at 39.5° is a consequence of the formation of aggregates of virus particles carrying neuraminic acid on their surface, and that the ts defect is in the neuraminidase but not the hemagglutinin molecule. It is postulated that neuraminidase is essential for the replication of influenza viruses and is required to remove neuraminic acid from the viral envelope to avoid aggregation of the progeny virus.     

Transfer of the hemagglutinin activity of influenza virus neuraminidase subtype N9 into an N2 neuraminidase background.

It has previously been shown that influenza virus neuraminidase (NA) of the N9 subtype is unusual in that it possesses hemagglutinin activity as well as NA activity. Loss of red cell binding in certain escape mutants suggested that the hemagglutinating site is separate from the NA active site and involves at least two of the polypeptide loops found on the surface of the molecule (Webster et al., 1987. J. Virol. 61, 2910-2916). We have used site-directed mutagenesis to transfer the amino acids in these loops at positions 368-370 and 399-403 of N9 NA (A/tern/Australia/G70c/75), separately and together, into subtype N2 NA (A/Tokyo/3/67). The three mutant proteins were expressed from an SV40 transient expression system (Fuerst et al., 1986. Proc. Natl. Acad. Sci. USA. 83, 8122-8126). The mutant which contained both loops of N9 NA had acquired the hemagglutinin activity of N9. The agglutinated red cells are released by the enzyme activity of N9 NA, indicating that the agglutination involves binding to sialic acid in the same configuration as does the parental N9 NA, and an inhibitor of NA did not affect hemagglutination, indicating that this site is separate from the NA site as in parental N9.     

H5N1型高致病性禽流感病毒血凝素蛋白及神经氨酸酶的B细胞抗原表位预测

目的预测H5N1亚型高致病性禽流感病毒HA蛋白和NA蛋白的B细胞表位,为基于B细胞表位的预防性疫苗设计提供依据。方法基于HA蛋白和NA蛋白的蛋白质序列,采用Kyte-Doolittle的亲水性方案,Emini方案,Karplus方案和Jameson-wolf抗原指数方案,并辅以MAGE蛋白的二级结构柔性区域分析,预测HA蛋白和NA蛋白的B细胞表位。结果分别预测出了6条血凝素蛋白(Hemagglutinin,HA)以及6条神经氨酸酶(Neuraminidase,NA)B细胞优势表位。结论这些B细胞表位可为禽流感疫苗的研制提供实验依据。     

Absence of neuraminidase from influenza C virus

Summary Influenza C viruses did not possess neuraminidase activity when examined using either fetuin or sialyllactose as substrate. Purified preparations of influenza C virus inhibited hemagglutination by NWS hemagglutinin. The hemagglutination inhibiting activity was a bolished by treatment of influenza C virus with neuraminidase. These findings indicated the absence of neuraminidase activity on influenza C virus particles.With 2 Figures