Antigenic drift in influenza A viruses. I. Selection and characterization of antigenic variants of A/PR/8/34 [HON1] influenza virus with monoclonal antibodies

Antigenic variants of A/PR/8/34 [HON1] influenza virus were selected after a single passage of the parent virus in embryonated chicken eggs in the presence of monoclonal antibodies to this virus. The monoclonal antibodies were produced by a hybridoma and were specific for an antigenic determinant on the HA molecule of the parent virus. Seven antigenic variants were analyzed with 95 monoclonal anti-HA antibodies prepared in vitro in the splenic fragment culture system. Three subgroups of antigenic variants were distinguished. The antigenic changes were primarily recognized by monoclonal antibodies to the strain- specific determinants of the parental hemagglutinin (HA) molecule. Monoclonal antibodies to HA determinants shared (in an identical or cross-reactive form) by parental virus and more than three heterologous viruses of the HON1 and H1N1 subtypes were unable to recognize the antigenic change on the variants. Similarly, heterogeneous antibody preparations could not differentiate between parental and variant viruses. The results are compatible with the idea that the HA of PR8 has available a large repertoire of antigenic modifications that may result from single amino acid substitutions, and that antigenic changes can occur in the strain- specific determinants on the HA molecule in the absence of concomitant changes in the cross-reactive HA determinants. The findings suggest that antigenic drift, in order to be epidemiologically significant, probably requires a series of amino acid substitutions in, or close to, the antigenic area on the HA molecule.     

Most influenza A virus-specific memory cytotoxic T lymphocytes react with antigenic epitopes associated with internal virus determinants

This paper shows that most murine (C57BL/6) influenza A virus-specific memory cytotoxic T lymphocyte (CTL) clones tested in limiting dilution did not react with the influenza A virus surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). This lysis of syngeneic target cells infected with the influenza A virus strains, Aichi (H3N2), PR8 (H1N1), or recombinant strain X31 (H3N2) indicates that most antigenic epitopes recognized are associated with internal virus determinants. X31 and PR8 share the internal, and X31 and Aichi the external, viral determinants. Extensive CTL cross-reactivity was observed in experiments with target cells infected with virus carrying internal determinants homologous with the priming virus. In contrast, when the internal viral determinants differed between the priming virus and the virus used to infect the target cells, and although HA and NA were homologous, we found almost complete CTL-specificity for the priming virus. Thus, the predominant reactivity of influenza A virus-specific CTL differs from that of anti-influenza A antibodies, which are primarily directed towards epitopes on the virus surface glycoproteins. This finding may be relevant for the role of influenza A virus-specific CTL in recurrent infections with different influenza A viruses.     

Idiotypy of clonal responses to influenza virus hemagglutinin

Anti-idiotype antisera were raised in syngeneic (BALB/c mice) and homologous (A/J mice) systems to study the cross-reactive idiotypes among monoclonal antibodies to PR8 and B/Lee virus HA and the expression of these idiotypes during primary and secondary antiviral responses of BALB/c mice. Extensive idiotypic cross-reactivity was demonstrated among monoclonal antibodies specific for distinct antigenic determinants on PR8 hemagglutinin (HA). The study of idiotypy of monoclonal antibodies against the same or overlapping antigenic determinants on B/Lee HA showed that these monoclonal antibodies may bear (a) a true individual idiotype not shared by other monoclonal antibodies, (b) idiotypes shared by few monoclonal antibodies, and (c) true cross-reactive idiotypes shared by all of these monoclonal antibodies. In contrast, no cross-reactive idiotypes were detectable among monoclonal antibodies to B/Lee HA and monoclonal antibodies to PR8 HA. Furthermore, we have shown that the anti-idiotype antibodies we used recognize determinants on monoclonal antibodies closely associated with antigenic binding sites. Finally, studies of the idiotypes expressed during primary and secondary antiviral HA responses of mice immunized with B/Lee virus revealed persistence of some idiotypes during both primary and secondary responses, whereas others were only expressed in the primary or secondary response.     

Preexisting human antibodies neutralize recently emerged H7N9 influenza strains

The emergence and seasonal persistence of pathogenic H7N9 influenza viruses in China have raised concerns about the pandemic potential of this strain, which, if realized, would have a substantial effect on global health and economies. H7N9 viruses are able to bind to human sialic acid receptors and are also able to develop resistance to neuraminidase inhibitors without a loss in fitness. It is not clear whether prior exposure to circulating human influenza viruses or influenza vaccination confers immunity to H7N9 strains. Here, we demonstrate that 3 of 83 H3 HA-reactive monoclonal antibodies generated by individuals that had previously undergone influenza A virus vaccination were able to neutralize H7N9 viruses and protect mice against homologous challenge. The H7N9-neutralizing antibodies bound to the HA stalk domain but exhibited a difference in their breadth of reactivity to different H7 influenza subtypes. Mapping viral escape mutations suggested that these antibodies bind at least two different epitopes on the stalk region. Together, these results indicate that these broadly neutralizing antibodies may contribute to the development of therapies against H7N9 strains and may also be effective against pathogenic H7 strains that emerge in the future.     

Active immunization against virus infections due to antigenic drift by induction of crossreactive cytotoxic T lymphocytes

We have examined whether active immunization with c13 protein, a hybrid protein of the first 81 amino acids of the viral NS1 nonstructural protein and the HA2 subunit of A/PR/8 (H1N1) hemagglutinin, could protect BALB/c mice from challenge with A/PR/8 H1 subtype virus. Mice immunized with the c13 protein had a significant reduction of pulmonary virus titers with A/PR/8 (H1) virus, but failed to limit the replication of A/PC (H3) virus, which reflects the in vitro CTL activity of c13 immune spleen cells. We observed that the epitope recognized by HA2 specific CTL, which are induced by a derivative of c13 protein, is highly conserved among H1 and H2 subtype virus strains. This led us to test whether active immunization with c13 protein would also limit pulmonary virus replication in mice infected with the A/TW virus, a virus of the H1 subtype, which was isolated in 1986, and with a virus of the H2 subtype, A/Japan/305/57. Immunized mice had significantly lower lung virus titers than did control mice, and did not possess any neutralizing antibodies to the challenger viruses. These results indicate that active immunization with a fusion protein containing the cross-reactive CTL epitope protects mice from influenza infection by inducing CTL against influenza A H1 and H2 subtype virus strains, which markedly vary in their antibody binding sites on the HA1. The ability to induce active cross-reactive immunization with a fusion protein which contains a highly conserved CTL epitope offers a model for vaccine approaches against viruses which undergo significant variations in their antibody binding sites.     

ANTIBODY RESPONSES TO ANTIGENIC DETERMINANTS OF INFLUENZA VIRUS HEMAGGLUTININ : I. Thymus Dependence of Antibody Formation and Thymus Independence of Immunological Memory

Using immunodiffusion methods it has been shown that purified hemagglutinin (HA) extracted from two related strains of influenza A viruses (A/PR8/34 and A/FM1/47) have two distinct antigenic determinants, or groups of determinants. One determinant is cross-reactive while the other is strain-specific. Antisera raised in normal mice against HA were shown to contain two populations of antibody molecules, each directed against one of the determinants. Immunization of thymus-deprived (TXBM) mice showed a strong thymus dependence of antibody formation to HA. However, the thymus dependence of antibody formation against the cross-reactive determinant could be overcome by repeated inoculations of HA in TXBM mice, indicating a different handling of two portions of the same molecule by the immunological system. Strong, secondary-type responses to the strain-specific determinant were observed in primed thymus-deprived mice after reconstitution with virgin thymus cells, showing that specific immunological memory was elicited by this determinant despite the absence of detectable antibody secretion. These findings are interpreted as examples of immunological recognition and memory mediated by B lymphocytes and discussed in terms of mechanisms of T and B lymphocyte co-operation. It is suggested that the helper effect of T lymphocytes is exerted at a late stage in the differentiation of specific populations of B cells into antibody-secreting cells.     

The Hemagglutinin Stem-Binding Monoclonal Antibody VIS410 Controls Influenza Virus-Induced Acute Respiratory Distress Syndrome

Most cases of severe influenza are associated with pulmonary complications, such as acute respiratory distress syndrome (ARDS), and no antiviral drugs of proven value for treating such complications are currently available. The use of monoclonal antibodies targeting the stem of the influenza virus surface hemagglutinin (HA) is a rapidly developing strategy for the control of viruses of multiple HA subtypes. However, the mechanisms of action of these antibodies are not fully understood, and their ability to mitigate severe complications of influenza has been poorly studied. We evaluated the effect of treatment with VIS410, a human monoclonal antibody targeting the HA stem region, on the development of ARDS in BALB/c mice after infection with influenza A(H7N9) viruses. Prophylactic administration of VIS410 resulted in the complete protection of mice against lethal A(H7N9) virus challenge. A single therapeutic dose of VIS410 given 24 h after virus inoculation resulted in dose-dependent protection of up to 100% of mice inoculated with neuraminidase inhibitor-susceptible or -resistant A(H7N9) viruses. Compared to the outcomes in mock-treated controls, a single administration of VIS410 improved viral clearance from the lungs, reduced virus spread in lungs in a dose-dependent manner, resulting in a lower lung injury score, reduced the extent of the alteration in lung vascular permeability and protein accumulation in bronchoalveolar lavage fluid, and improved lung physiologic function. Thus, antibodies targeting the HA stem can reduce the severity of ARDS and show promise as agents for controlling pulmonary complications in influenza.     

QUALITATIVE DIFFERENCES IN THE ANTIGENIC COMPOSITION OF INFLUENZA A VIRUS STRAINS

A study of the PR8, Christie, Talmey, W.S., and swine strains of influenza A virus by means of antibody absorption tests revealed the following findings: 1. Serum antibody could be specifically absorbed with allantoic fluid containing influenza virus or, more effectively, with concentrated suspensions of virus obtained from allantoic fluid by high-speed centrifugation or by the red cell adsorption and elution technique. Normal allantoic fluid, or the centrifugalized sediment therefrom, failed to absorb antibodies. Influenza B virus (Lee) caused no detectable absorption of antibody from antisera directed against influenza A virus strains, but it specifically absorbed antibody from Lee antisera. 2. The neutralizing, agglutination-inhibiting, and complement-fixing anti-bodies in ferret antisera were completely absorbed only by the homologous virus strain, even though 2 absorptions were carried out with large amounts of heterologous virus strains. 3. PR8 virus appeared to have the broadest range of specific antigenic components for it completely absorbed the heterologous antibodies in Christie and W.S. antisera and left only those antibodies which reacted with the respective homologous strains. The other virus strains (Christie, Talmey, W.S., swine) were more specific in the absorption of heterologous antibodies and completely removed only those antibodies which reacted with the absorbing virus. 4. The absorption tests revealed a higher degree of specificity and individuality of the virus strains than the various cross reactions previously reported. The strain specificity of PR8 virus was equally manifest in absorption tests with ferret sera and with human sera following vaccination. 5. The amount of homologous antibody remaining in a PR8 ferret serum after absorption with PR8 virus, obtained by the red cell adsorption and elution method, varied inversely as the concentration of virus used for absorption. A given concentration of virus, however, absorbed a greater percentage of neutralizing antibodies than either agglutination-inhibiting or complement-fixing antibodies.     

The diversity of the influenza-specific primary B-cell repertoire in BALB/c mice

The primary immune response of BALB/c mice to influenza (PR8) hemagglutinin (HA), a complex protein antigen, has been examined by the splenic focus assay, and the resulting monoclonal anti-HA antibodies have been characterized by their reactivity with heterologous viruses. The analysis of the primary B-cell response to HA revealed marked differences from responses previously defined for haptenic determinants. There were following differences: (a) the frequency of HA-specific B cells in both conventional and germ-free BALB/c mice was 1 in 1.0-1.5 X 10(5) splenic B cells, which is substantially lower than the frequency of B cells responsive to various simple haptenic determinants; (b) monoclonal anti-HA antibodies were predominantly of the IgA or IgM isotypes instead of IgG, which dominates antihapten responses; and (c) after immunization, the frequency of anti-HA-specific B cells increases by 10- to 50-fold, which is much greater increase than that observed after immunization with haptenic determinants. Fine specificity analysis of primary monoclonal HA-specific antibodies revealed extensive diversity and a considerable overlap with the specificities obtained from immune mice. Given the low overall frequency of HA-specific B cells, it could be calculated that the representation of most HA-specific clonotypes within the B-cell repertoire could not exceed 1 in 10(7) B cells. These findings indicate that the primary B-cell clonotype repertoire is extremely diverse and largely antigen independent in its generation.     

Mouse H-2k-restricted cytotoxic T cells recognize antigenic determinants in both the HA1 and HA2 subunits of the influenza A/PR/8/34 hemagglutinin

We have constructed two chimeric influenza hemagglutinin (HA) genes in which the HA1 and HA2 subunits of the HA molecule have been interchanged between influenza A/PR/8/34 (H1 subtype) and A/NT/60/68 (H3 subtype). These genes were used to construct recombinant vaccinia viruses that expressed intact chimeric HA. These recombinant viruses were used to test whether murine CTL recognize antigenic determinants in either the HA1, HA2, or both subunits. We found that both subunits of the HA molecule contain determinants for CTL. This implies that CTL have, at least in part, separate antigenic determinants from B lymphocytes, which recognize mainly epitopes within the HA1 subunit.     

Participation of the major histocompatibility complex in antibody recognition of viral antigens expressed on infected cells

The capacity of the antibody repertoire to recognize complex antigens on viral-infected cells was investigated at the level of monoclonal B cell responses. A majority of primary B cells responsive to PR8(H1N1)-infected H-2 syngeneic cells produced antibody that bound viral determinants only in the context of infected cells and not the isolated virion. An examination of the fine specificity of such antibodies revealed that most could be distinguished by a panel of cells infected with closely related heterologous H1 influenza strains. Indeed, most antibodies bound hemagglutinin determinants of PR8 exclusively, and few were broadly cross-reactive. An examination of the same antibodies for their recognition of cell surface antigens revealed that the majority recognized MHC-encoded antigenic determinants. Thus, most BALB.K (H-2k) primary B cells responsive to PR8-L919 (H-2k) cells produced monoclonal antibodies that bound PR8-antigens only in the context of H-2Dk-infected cells. Most C57BL/10 (H-2b) B cells responsive to PR8-EL4 (H-2b) cells produced monoclonal antibodies that bound PR8 antigens only in the context of H-2Kb-infected cells. These latter antibodies were further shown to recognize that H-2Kb molecule by virtue of their capacity to be discriminated by a panel of PR8-infected H-2Kb mutant cells. These findings demonstrate that much of the antibody repertoire is capable of highly specific complex recognition of viral antigenic determinants in the context of the appropriate MHC alloantigen.     

A Trivalent Virus-Like Particle Vaccine Elicits Protective Immune Responses against Seasonal Influenza Strains in Mice and Ferrets

There is need for improved human influenza vaccines, particularly for older adults who are at greatest risk for severe disease, as well as to address the continuous antigenic drift within circulating human subtypes of influenza virus. We have engineered an influenza virus-like particle (VLP) as a new generation vaccine candidate purified from the supernatants of Sf9 insect cells following infection by recombinant baculoviruses to express three influenza virus proteins, hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1). In this study, a seasonal trivalent VLP vaccine (TVV) formulation, composed of influenza A H1N1 and H3N2 and influenza B VLPs, was evaluated in mice and ferrets for the ability to elicit antigen-specific immune responses. Animals vaccinated with the TVV formulation had hemagglutination-inhibition (HAI) antibody titers against all three homologous influenza virus strains, as well as HAI antibodies against a panel of heterologous influenza viruses. HAI titers elicited by the TVV were statistically similar to HAI titers elicited in animals vaccinated with the corresponding monovalent VLP. Mice vaccinated with the TVV had higher level of influenza specific CD8+ T cell responses than a commercial trivalent inactivated vaccine (TIV). Ferrets vaccinated with the highest dose of the VLP vaccine and then challenged with the homologous H3N2 virus had the lowest titers of replicating virus in nasal washes and showed no signs of disease. Overall, a trivalent VLP vaccine elicits a broad array of immunity and can protect against influenza virus challenge.     

Influenza virus site recognized by a murine helper T cell specific for H1 strains. Localization to a nine amino acid sequence in the hemagglutinin molecule

The functional helper T cell line Vir-2, derived from a PR8 (H1N1) influenza virus-immunized BALB/c mouse, proliferates in response to syngeneic antigen-presenting cells and naturally occurring strains of subtype H1 human influenza virus from 1934-1957 and 1977-1980 isolates. A conserved region of the hemagglutinin molecule around amino acid position 115 in the heavy chain (HA1) was implicated as being important in this recognition by the lack of stimulatory activity associated with a glutamic acid to lysine substitution at position 115 in the laboratory mutant RV6, derived from wild-type PR8. Characterization of the stimulatory determinant on the wild-type hemagglutinin molecule was then undertaken using cleavage products and synthetic peptides. Vir-2 cells recognized the reduced and alkylated purified HA1 of PR8 virus, and this reactivity was retained after cleavage at methionine and tryptophan residues. High-pressure liquid chromatography separation of cleavage fragments indicated that a short sequence of the HA1 containing residue 115 was being recognized. This recognition was localized to a nine amino acid segment (positions 111-119) by assaying stimulation with synthetic peptide homologues of different lengths from that region. As with native hemagglutinin, Vir-2 cells responded to active peptides when presented by H-2d but not H-2k antigen-presenting cells.     

Antigenic variants of influenza A virus (PR8 strain) III. Serological relationships of a line of variants derived in sequence in mice given homologous vaccine

Seven variant strains of influenza A PR8-S virus, each derived from the previous one by serial passage in the lungs of mice immunized with the homologous agent have been produced. With the H.I. and neutralization procedures these variants showed a progressive serological deviation from the parent PR8-S virus. The seven variants provoked antibodies in varying titers to the preceding variants and the parent virus but not in relation to their position in the series. Thus, the seventh variant provoked significantly more antibody to the PR8-S virus than did the fifth variant. A possible explanation for this is presented. The first four variant viruses showed progressively less ability to react with antisera of the preceding variants and the PR8-S virus, and the three most recently derived variants showed essentially no ability to react with PR8-S and first variant antisera. The variant viruses remained antigenically stable through numerous lung passages in normal mice. Cross absorption tests revealed common antigenic components among the variant viruses and also individual characteristics which classify them as being different from one another. The implications of these findings in relation to studies by others have been discussed.     

Antigenic variants of influenza A virus,PR8 strain. I. Their development during serial passage in the lungs of partially immune mice

Antigenically different strains of mouse-adapted PR8 influenza A virus have been produced by 17 serial passages of the virus in the lungs of mice immunized with the homologous agent. Comparative serological tests show that the variant strains share antigenic components with the parent strain but the dominant antigen is different. By means of antibody absorption it was shown that the "new" antigenic component of the variant was already present in minor amounts up to the eighth passage and thereafter gained prominence with continued passage in vaccinated mice. Groups of mice vaccinated with either the PR8-S or T₂₁ virus and having comparable antibody titers showed no growth of virus in the lungs following aid-borne challenge with homologous strains. On the other hand, following heterologous air-borne challenge no deaths occurred, but virus grew in the lungs of both groups of vaccinated mice. Almost unrestricted virus multiplication took place in the lungs of mice vaccinated with the parent strain and challenged with the PR8-T₂₁ virus which resulted in extensive consolidation. Less virus grew in the lungs of the mice vaccinated with the variant strains and challenged with the PR8-S virus. In these animals only microscopic evidence of changes due to virus growth in the lungs was observed. The successful serial passage of PR8 influenza A virus in immunized animals was dependent on the initial selection of mice with uniformly low H.I. antibody titers as determined on tail blood, and the intranasal instillation of sufficient virus to favor the survival of those virus particles least related to the antibodies present. The epidemiological implications of these observations are discussed briefly.     

GENETIC STUDIES OF INFLUENZA VIRUSES : I. VIRAL MORPHOLOGY AND GROWTH CAPACITY AS EXCHANGEABLE GENETIC TRAITS. RAPID IN OVO ADAPTATION OF EARLY PASSAGE ASIAN STRAIN ISOLATES BY COMBINATION WITH PR8

The passage of newly isolated, filamentous Asian (A2) influenza viruses in the presence of non-infective PR8 (A) virus results in the rapid emergence of virus of Asian (A2) antigenicity but PRS-like growth capacity and spherical morphology. Evidence is presented that this effect results from genetic interaction of the infective Asian and non-infective PR8 viruses rather than from spontaneous change of the Asian strain. It is concluded that influenza viral morphology, growth rate and growth capacity are associated genetic traits which distinguish unadapted from adapted strains, and which are transferable by recombination. A pragmatic consequence of these experiments is the fact that conditions have been defined for the rapid adaptation of early passage influenza virus isolates to the chick embryo allantoic sac. Such adaptation is attended by an increase in viral yield which has obvious implications for vaccine production during future epidemics with new antigenic types.     

A serologic recapitulation of past experiences with influenza A; antibody response to monovalent vaccine

The results of the present study provide a striking demonstration of antibody orientation produced by the dominant antigens of the strains of influenza virus encountered at childhood. The homologous and heterologous antibody response to monovalent vaccines containing swine, PR8, FM1, or Cuppett viruses show that the antibody-forming mechanisms of children born after 1943 are oriented to strains of influenza A-prime; of recruits to strains of influenza A; and of persons over 30 years of age to a strain of swine influenza. It was shown that antibody response to heterotypic viruses appears to provide a serologic index of the amount of experience each of the segments of the general population studied have had with antigenic variants of influenza virus. Finally it was demonstrated that the antibody response of humans to strains of influenza A or influenza A-prime is remarkably uniform for isolates of each antigenic type.     

Development of competitive enzyme immunoassay for the detection of influenza A(H5) virus antibodies

An indirect enzyme immunoassay (EIA) using the purified fraction of surface viral glycoproteins (GP) as an antigen for solid phase sensitization was not shown to be a specific method for the differential detection of influenza A(HS) (HS-Ab) virus antibodies (Abs) due to total conservative epitopes in the structure of GPs of influenza A(H5) and A(H1NI) viruses. The cross activity of some monoclonal Abs (MAbs) to influenza A(H5) and A(HIN1) viruses, which had been obtained at the Research Institute of Influenza, was proof of the presence of total immunodominant determinants in the structure of influenza H1 and H5 virus hemagglutinin (HA). In this connection, an EIA, which was based on the competition of influenza A(H5) H5-Ab virus HA-specific MAbs in the test sera for an association with influenza A(H5) virus, was proposed for the subtype-specific detection of H5 Ab. Comparison of the results of competitive EIA (cEIA), microneutralization (MN) test and HA inhibition test (HAIT) (using equine red blood cells) in the examination of sera obtained from 44 volunteers immunized with inactivated vaccine containing influenza A/Indonesia/5/2005 (H5N1) virus showed the high sensitivity and specificity of cEIA in detecting H5-specific Abs. The effectiveness of cEIA for the sera strictly positive for the content of H5 Abs was close to that of MN test and was 9-34% higher than HAIT (depending on those used in the analysis of H5 virus antigens). cEIA may be proposed to assess new influenza vaccines as an additional laboratory test. Since the infectious virus is not used during cEIA, it may be recommended for the serodiagnosis of influenza A(H5) at practical virological laboratories.     

AMINO ACID COMPOSITION OF HIGHLY PURIFIED VIRAL PARTICLES OF INFLUENZA A AND B

Microbiological assays for amino acids were made on hydrolysates of four to five highly purified preparations each of influenza A virus (PR8 strain) and influenza B virus (Lee strain). The results of the assays indicated that these strains of influenza virus contain approximately the same amounts of alanine, aspartic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, and valine. However, significant differences were found in the values for arginine, glutamic acid, lysine, tryptophane, and tyrosine. It is believed that these differences may provide, at least in part, a chemical explanation for some of the differing properties of the PR8 and Lee strains of influenza viruses.     

ANTIGENIC RELATIONSHIP OF BRITISH SWINE INFLUENZA STRAINS TO STANDARD HUMAN AND SWINE INFLUENZA VIRUSES : THE USE OF CHICKEN AND FERRET ANTISERA IN RED CELL AGGLUTINATION

The antigenic relationships of Type A (PR 8, WS) and Type B (Lee) human strains and the Shope and British (Cambridge, North Ireland) swine strains were studied by specific antiserum inhibition of chicken red cell agglutination by the influenza virus. The Cambridge and North Ireland strains were found to be closely related to the Type A strains and differentiated from the Shope virus. The distinctive antigenicity of the Lee strain of Type B was confirmed. Specific antibodies were developed in chickens following single intraperitoneal injections of influenza virus. Inhibition tests yielded results, in the antigenic analysis of the influenza viruses examined, comparable to those obtained with ferret antisera. Specific inhibition of hemagglutination by influenza virus proved an effective method for the study of strain relationships.