Characterization of avian influenza viruses. Designation of a newly recognized haemagglutinin.

Studies with specific antisera to the haemagglutinin and neuraminidase antigens of all the influenza A subtypes show that A/turkey/Wisconsin/66 influenza virus, originally included in the Hav6 subtype, does not react in either haemagglutinin inhibition or immunodiffusion tests with antisera to Hav6. It is therefore proposed that A/turkey/Wisconsin/66 be placed in a new subtype designated Hav9. The neuraminidase antigens of the Hav6 subtype were further characterized and were shown to be N1, N2, Neq2, and Nav5 subtypes. Hav6 influenza viruses isolated from turkeys over an 11-year period showed little antigenic drift. The haemagglutinin and neuraminidase of A/shearwater/Australia/72 (Hav6Nav5) were identical with those of a virus isolated 8 years previously from a turkey in California: A/turkey/California/64 (Hav6Nav5).     

Isolation and characterization of influenza A viruses from avian species in Hong Kong.

Surveillance of apparently healthy ducks, chickens, and geese at a poultry dressing plant in Hong Kong yielded 51 haemagglutinating viruses 25 of which were influenza A viruses. Of these, 24 were subtyped into 13 combinations based on haemagglutinin and neuraminidase surface antigens. Of the 13 different influenza A viruses isolated, 7 possessed combinations of haemagglutinin and neuraminidase subunits that have not been reported previously—i.e., Hav2N1, Hav2Nav5, Hav4N2, Hav7N2, Hav7Nav1, Hav7Nav3, and Hav7Nav6. Four of the isolates were non-avid: they were not neutralized by antisera to any of the reference subtypes of influenza A viruses, yet antisera to each isolate inhibited both that virus and a known reference strain. The large number of combinations of haemagglutinin and neuraminidase and the isolation of two different influenza A viruses from one duck suggests that recombination may be occurring in nature.     

Antibodies to human influenzavirus neuraminidase (the A-Asian-57 H2N2 strain) in sera from Australian pelagic birds

Sera collected from Australian pelagic birds specifically inhibited the neuraminidase of the Asian/57 (H2N2) strain of human influenzavirus. Neuraminidase inhibition titres of some sera were high and the avidity of the inhibitor was comparable to that of specific antibody. The neuraminidase of A/Hong Kong/1/68 (H3N2), which has undergone considerable antigenic drift in man since 1957, was inhibited to a lesser extent by the bird sera, while the neuraminidases of the A/BEL/42 (H0N1) and A/FM/1/47 (H1N1) strains were not inhibited at all. The inhibitor could be removed from the sera by adsorption with A/57 virus particles, but not by particles of A/BEL or A/FM1 viruses. These results suggested that the inhibitor in the bird sera was specific antibody. The antibodies to A/57 neuraminidase were found in sera from wedge-tailed shearwaters (Puffinus pacificus) and noddy terns (Anous minutus) nesting on islands off the north-east coast of Australia. They were not found in sera from bridled terns (Sterna anaetheta) or brown gannets (Sula leucogaster) nesting on the same islands. Antibodies to A/57 neuraminidase were also detected in sera from short-tailed shearwaters (Puffinus tenuirostris), which migrate around the Pacific Ocean, suggesting that these birds may be responsible for bringing avian influenzaviruses from areas in the Northern Hemisphere into Australian coastal waters.     

Some observations on the circulation of influenzaviruses in domestic and wild birds

Four viruses isolated from poultry in the USSR and Poland were identified as influenza A strains. One strain was closely related to fowl plague virus, the second showed an antigenic relationship to A/chicken/Scotland/59 (Hav5N1), and two others were antigenically related to A/duck/Ukraine/1/63 (Hav7Neq2) and A/duck/England/56 (Hav3Nav1). Antibodies to different strains of influenzavirus were detected in sera collected from poultry and from wild birds in the northern area of the USSR. More of the sera collected from migratory sea birds in the autumn gave HI reactions than did those collected in the spring. HI reactions were of higher titre when recent locally isolated viruses were used than with reference strains of influenzavirus. Sera collected in the autumn of 1969 from wild sea birds (mostly herring gulls, ducks, arctic loons, and long-tailed ducks) frequently showed HI activity not only with avian influenzavirus but also with A/equine/Miami/1/63 (Heq2 Neq2) and A/Hong Kong/1/68 (H3N2). The significance of these findings is discussed.     

A new subtype of avian influenzavirus: antigenic characteristics of envelope antigens

The haemagglutination and neuraminidase antigens of three influenza A isolates from ducks in the Ukraine were compared with those of a collection of reference strains of influenza A virus. Duck/Ukraine/1/60 virus contained haemagglutinin related to that of duck/England/56 while its neuraminidase was related to that of turkey/Wisconsin/68 virus and the human A/Hong Kong/1/68 virus. Duck/Ukraine/2/60 and duck/Ukraine/1/63 were themselves closely related. They contained haemagglutinin antigens unrelated to the six haemagglutinin subtypes previously described for avian influenzaviruses and it is suggested that they should be classified as belonging to haemagglutinin subtype Hav7. The neuraminidase antigens of these isolates were antigenically related to those of a number of other avian influenza viruses isolated in England, Canada, and Italy and to that of A/equine/ Miami/63 virus.     

Characterization of a novel haemagglutinin subtype (H13) of influenza A viruses from gulls

Influenza A virus strains isolated in the United States of America from ring-billed and Franklin gulls (Larus delawarensis, L. pipixcan) were found to possess a haemagglutinin (HA) antigen distinct from those of the twelve previously designated haemagglutinin subtypes of influenza A virus. Serological assays with antisera to reference strains representing the HA subtypes 1-12 and to a gull isolate, A/gull/Maryland/704/77, showed that the haemagglutinin of the gull virus was not related antigenically to the previously designated subtypes. In addition, comparison of the nucleotide sequences, and deduced amino acid sequences, of the 3′ region of the RNA genes coding for haemagglutinin indicated that the gull viruses represent a genetically distinct group. We propose that this new HA antigen, which has so far been detected only in gulls, be designated H13 and that A/gull/Maryland/704/77 (H13N6) be designated the reference strain for this subtype.     

Diversity of influenza A virus subtypes isolated from domestic poultry in Hong Kong.

The second phase of a 2-year influenza virus surveillance programme of domestic avian species in Hong Kong (up to October 1977) yielded influenza A virus, Newcastle disease virus, and Hong Kong paramyxovirus, as well as unidentified haemagglutinating agents. These viruses were isolated from the trachea or cloaca of apparently healthy domestic ducks, geese, and chickens originating from China and Hong Kong. Twenty-five combinations of haemagglutinin and neuraminidase surface antigens were identified from the 136 influenza A viruses isolated. Eight of the combinations do not appear to have been previously reported — Hav3Nav2, Hav4Nav2, Hav4Nav4, Hav4Nav5, Hav4Neq1, Hav6Nav4, Hav6Nav6, and Hav9Nav1. The existence of such a diverse pool of influenza virus genetic information may play a role in the emergence of new human pandemic strains.     

A new avian influenza virus from feral birds in the USSR: Recombination in nature?

Six avian influenza A viruses isolated in the USSR were characterized antigenically by using specific antisera to the isolated surface subunits of the known reference strains. Three of the viruses, all isolated from the same region, were characterized as A/duck/Ukraine/63 (Hav7 Neq2), and a virus isolated from a crow was of the Hong Kong/68 (H3 N2) type. The remaining two viruses were novel in that they possessed Hav7 Nav2 antigens, a combination that has not previously been reported. It is suggested that these new influenza viruses might have arisen by recombination in nature between the A/duck/Ukraine/63 (Hav7 Neq2) and A/tern/So. Africa/61 (Hav5 Nav2) strains of avian influenza viruses.     

Studies on relationships between human and porcine influenza. 2. Immunological comparisons of human A-Hong Kong-68 virus with influenza A viruses of porcine origin

Antigenic comparisons were made between the human A/Hong Kong/68 (H3N2) virus and a collection of influenza A viruses of swine origin. Haemagglutination-inhibition and neuraminidase-inhibition tests were used in addition to immunoprecipitin tests with monospecific antisera prepared against purified haemagglutinin and neuraminidase preparations. The antigenic relationships revealed by the studies are summarized as follows: (1) swine/Taiwan/7310/70 virus contained envelope antigens that were antigenically indistinguishable from those of A/Hong Kong/68 virus, (2) “classical” strains of swine influenzavirus related to A/swine/Iowa/15/30 (Hsw1N1) isolated between 1930 and 1967 contained neuraminidase that was antigenically distinct from that of A/Hong Kong/68 virus but related to that of human A0 and A1 viruses, and (3) the haemagglutinins of certain strains of “classical” influenza A virus appeared to show a minor antigenic relationship with the haemagglutinin of A/Hong Kong/68 virus. Immunoprecipitin tests suggested that this relationship was confined to only one of the two antigenic components of the haemagglutinin subunit. The antigenic relationships are discussed in respect of possible epidemiological relationships between human and swine influenza A viruses. It is proposed that the swine/Taiwan isolates be designated A/swine/Taiwan/70 (H3N2), indicating their antigenic identity with the human A/Hong Kong/1/68 virus.     

Recent zoonoses caused by influenza A viruses

Influenza is a highly contagious, acute illness which has afflicted humans and animals since ancient times. Influenza viruses are part of the Orthomyxoviridae family and are grouped into types A, B and C according to antigenic characteristics of the core proteins. Influenza A viruses infect a large variety of animal species, including humans, pigs, horses, sea mammals and birds, occasionally producing devastating pandemics in humans, such as in 1918, when over twenty million deaths occurred world-wide. The two surface glycoproteins of the virus, haemagglutinin (HA) and neuraminidase (NA), are the most important antigens for inducing protective immunity in the host and therefore show the greatest variation. For influenza A viruses, fifteen antigenically distinct HA subtypes and nine NA subtypes are recognised at present; a virus possesses one HA and one NA subtype, apparently in any combination. Although viruses of relatively few subtype combinations have been isolated from mammalian species, all subtypes, in most combinations, have been isolated from birds. In the 20th Century, the sudden emergence of antigenically different strains in humans, termed antigenic shift, has occurred on four occasions, as follows, in 1918 (H1N1), 1957 (H2N2), 1968 (H3N2) and 1977 (H1N1), each resulting in a pandemic. Frequent epidemics have occurred between the pandemics as a result of gradual antigenic change in the prevalent virus, termed antigenic drift. Currently, epidemics occur throughout the world in the human population due to infection with influenza A viruses of subtypes H1N1 and H3N2 or with influenza B virus. The impact of these epidemics is most effectively measured by monitoring excess mortality due to pneumonia and influenza. Phylogenetic studies suggest that aquatic birds could be the source of all influenza A viruses in other species. Human pandemic strains are thought to have emerged through one of the following three mechanisms: genetic reassortment (occurring as a result of the segmented genome of the virus) of avian and human influenza A viruses infecting the same host direct transfer of whole virus from another species the re-emergence of a virus which may have caused an epidemic many years earlier. Since 1996, the viruses H7N7, H5N1 and H9N2 have been transmitted from birds to humans but have apparently failed to spread in the human population. Such incidents are rare, but transmission between humans and other animals has also been demonstrated. This has led to the suggestion that the proposed reassortment of human and avian viruses occurs in an intermediate animal with subsequent transference to the human population. Pigs have been considered the leading contender for the role of intermediary because these animals may serve as hosts for productive infections of both avian and human viruses and, in addition, the evidence strongly suggests that pigs have been involved in interspecies transmission of influenza viruses, particularly the spread of H1N1 viruses to humans. Global surveillance of influenza is maintained by a network of laboratories sponsored by the World Health Organization. The main control measure for influenza in human populations is immunoprophylaxis, aimed at the epidemics occurring between pandemics.     

Antigenic relationships between type a influenza viruses of human, porcine, equine, and avian origin

This paper summarizes the available information on the relationship of two envelope antigens (haemagglutinin and neuraminidase) of influenzaviruses isolated from different hosts. The relationship of the haemagglutinin antigens was based on the results of haemagglutination inhibition tests with postinfection sera and that of the neuraminidase antigens on the results of neuraminidase inhibition and gel precipitation tests with hyperimmune and monospecific sera. On the basis of the antigenic specificity of the haemagglutinin, the influenzaviruses of human origin are divided into several subtypes (H0, H1, H2); viruses of equine origin could be divided into two subtypes (Heq1, Heq2). Porcine influenza strains are regarded as belonging to a single subtype, all of them being related to the prototype swine influenzavirus A (swine/Iowa/15/30). Within the avian influenzaviruses, 6 antigenic subtypes were described in earlier studies. Antigenic relationships between the haemagglutinin of strains from different hosts were infrequent but were demonstrated and confirmed between human A/Hong Kong/68 and equine viruses and between A/Hong Kong/68 and swine/Taiwan/69. The swine/Taiwan/69 virus also shared the related neuraminidase with A/Hong Kong/68 virus, and represents the only isolation from nonhuman sources of an influenzavirus identical with a human pandemic strain. The studies on the antigenic specificity of the neuraminidases demonstrated 8 antigenic varieties of neuraminidase among avian influenzaviruses and also that the neuraminidase grouping did not correspond with the antigenic grouping with regard to haemagglutinin. The relationships between human and nonhuman influenzaviruses are emphasized because of their significance to studies on the origin of influenza pandemics in man.     

Frequency of naturally occurring antibody to influenza virus antigenic variants selected in vitro with monoclonal antibody.

Antigenic variants of A/Texas/77 (H3N2) virus were selected in vitro using monoclonal antibody to virus haemagglutinin (HA). The antigenic variants and parental A/Texas/77 viruses were used to to evaluate the frequency of anti-HA antibodies in the sera of children and adults using single-radial-haemolysis (SRH) tests. Twenty to 41% of selected sera from adults, which contained antibody to the parental A/Texas/77 virus, failed to react with the different antigenic mutant viruses. A higher proportion of sera from children (37-58%) failed to react with the antigenic variants. Certain human sera and particularly those of children would appear to possess a more limited antibody repertoire to influenza HA, potentially allowing new antigenic variants to escape neutralization and spread in the community.     

The origin of pandemic influenza

Serological tests, using antisera specific to the surface subunits of the viruses (e.g., antisera to the haemagglutinin subunits devoid of any antineuraminidase or anti-host antigen activity) showed that the haemagglutinin subunits of the Hong Kong virus were, immunologically, distinct from the subunits of the preceding A/Asian strains. On the other hand, the neuraminidase subunits of the Hong Kong virus are related to those of the A/Asian strains. The haemagglutinin subunits of influenzaviruses are composed of two pairs of light and heavy polypeptide chains and it was found that the amino acid sequence of the light and heavy chains from the haemagglutinin subunits of the Hong Kong virus differed remarkably from those of the preceding A/Asian strains. These findings suggest that Hong Kong virus did not arise by mutation from a pre-existing human strain and that it probably arose by the selection of a genetic recombinant in a partially immune population. It is postulated that the recombinant was formed as the result of the mixed infection of an animal or bird with an animal or avian influenzavirus and a human A/Asian strain. The animal (or avian) virus could have donated the haemagglutinin subunits of A/Hong Kong/1/68 virus and the neuraminidase subunits could have come from the human A/Asian strain.     

Antibodies to influenza viruses (including the human A2-Asian-57 strain) in sera from Australian shearwaters (Puffinus pacificus)

Sera were collected from 320 shearwaters (Puffinus pacificus chlororhynchus) nesting on two islands off the east coast of northern Australia in December 1969. About 10% of these sera specifically inhibited the neuraminidase of the 1957 strain of human influenza (A2/Asian/57), some to high titre.     

Supplementation of conventional trivalent influenza vaccine with purified viral N1 and N2 neuraminidases induces a balanced immune response without antigenic competition

Influenza viruses neuraminidase (NA) were chromatographically extracted from influenza viruses A/Nanchang/933/95 H3(NC)N2(NC) [R] and A/Johannesburg/82/96 H1(JH)N1(JH) [R] and used to supplement conventional inactivated trivalent influenza vaccine. Immunization of mice with this preparation resulted in high titers of antibodies to both hemagglutinins (HA) and neuraminidases (NA); there were no significant differences in the anti-HA antibody titers between the conventional and the supplemented vaccine preparation. Likewise, there were no significant differences in anti-NA antibody titers between the supplemented vaccine and titers from mice immunized with a neuraminidase vaccine containing a mixture of N1-NA and N2-NA. There was no evidence of a diminution of the immune response to the HA components of the vaccine despite the presence of antigenically equivalent amounts of both N1-NA and N2-NAs. Homotypic and distantly related heterotypic infections for both H1, N1 and H3N2 subtypes were suppressed and greater reduction in pulmonary virus titers (PVT) were observed in the trivalent vaccine supplemented with purified neuraminidase from each subtype, N1 and N2. Effects on the influenza B viral components were not studied. Previous studies on supplementation of conventional influenza vaccine focused only on monovalent H3N2 vaccine preparations; this study demonstrates in a mouse model system that supplementation of trivalent influenza vaccine with both influenza A subtype neuraminidases, N1 and N2 is highly immunogenic for HA and NA of each subtype and efficacious in protecting against influenza from homotypic and heterotypic infectious challenges of either subtype.     

Generation and evaluation of a high-growth reassortant H9N2 influenza A virus as a pandemic vaccine candidate

H9N2 subtype avian influenza viruses (AIVs) are widely distributed in avian species and were isolated from humans in Hong Kong and Guangdong province, China in 1999 raising concern of their potential for pandemic spread. We generated a high-growth reassortant virus (G9/PR8) that contains the hemagglutinin (HA) and neuraminidase (NA) genes from the H9N2 avian influenza virus A/chicken/Hong Kong/G9/97 (G9) and six internal genes from A/Puerto Rico/8/34 (PR8) by genetic reassortment, for evaluation as a potential vaccine candidate in humans. Pathogenicity studies showed that the G9/PR8 reassortant was not highly pathogenic for mice or chickens. Two doses of a formalin-inactivated G9/PR8 virus vaccine induced hemagglutination inhibiting antibodies and conferred complete protection against challenge with G9 and the antigenically distinct H9N2 A/Hong Kong/1073/99 (G1-like) virus in a mouse model. These results indicate that the high growth G9/PR8 reassortant has properties that are desirable in a vaccine seed virus and is suitable for evaluation in humans for use in the event of an H9 pandemic.     

Serological studies with influenza A(H1N1) viruses cultivated in eggs or in a canine kidney cell line (MDCK)

Pairs of influenza A(H1N1) viruses cultivated from the same clinical specimen in canine kidney (MDCK) cells or in embryonated hens'' eggs can frequently be distinguished by their reactions with monoclonal antibodies to haemagglutinin and with antibodies in ferret or human sera. Egg-adapted virus, further passaged in MDCK cultures remained ”egg-like” in serological characteristics indicating that the differences in their serological reactions were not a direct result of host cell-dependent glycosylation of the haemagglutinin. Haemagglutination-inhibiting (HI) or virus neutralizing antibodies in human sera can be detected more frequently, and to higher titre, in tests employing virus grown exclusively in MDCK cells than in tests with virus adapted to growth in embryonated eggs. Striking differences were detected in the serological reactions in HI tests when sera from ferrets infected with egg-grown virus were tested against a series of strains of influenza A(H1N1) virus isolated in 1983 and adapted to growth in eggs. In contrast, sera from ferrets infected with MDCK-derived virus failed to distinguish serologically between the same viruses that had been passaged exclusively in MDCK cells and also revealed relatively small differences between their egg-adapted counterparts.     

Antigenic and amino acid sequence analysis of the variants of H1N1 influenza virus in 1986

Since their reintroduction to human populations in 1977, influenza A viruses of the H1N1 subtype have undergone antigenic drift. Recently a distinct antigenic variant, A/Singapore/6/86, has been almost exclusively isolated internationally, and the antigenic properties and amino acid sequence of its haemagglutinin have been determined and compared with those of the haemagglutinins of other H1N1 viruses, in particular A/Chile/1/83. Fourteen amino acid sequence differences are detected between the HA1 components of these two viruses, ten of which are different from equivalent residues in the haemagglutinins of all H1N1 viruses isolated between 1982 and 1983, and seven of which are novel in the haemagglutinins of all H1N1 viruses sequenced to date. The results are discussed in relation to the three-dimensional structure of the haemagglutinin and the location of the previously defined antigenically important regions.     

Predominance of influenza virus A(H3N2) 3C.2a1b and A(H1N1)pdm09 6B.1A5A genetic subclades in the WHO European Region, 2018–2019

BackgroundThe 2018/2019 influenza season in the WHO European Region was dominated by influenza A (H1N1)pdm09 and (H3N2) viruses, with very few influenza B viruses detected.MethodsCountries in the European Region reported virus characterization data to The European Surveillance System for weeks 40/2018 to 20/2019. These virus antigenic and genetic characterization and haemagglutinin (HA) sequence data were analysed to describe and assess circulating viruses relative to the 2018/2019 vaccine virus components for the northern hemisphere.ResultsThirty countries reported 4776 viruses characterized genetically and 3311 viruses antigenically. All genetically characterized A(H1N1)pdm09 viruses fell in subclade 6B.1A, of which 90% carried the amino acid substitution S183P in the HA gene. Antigenic data indicated that circulating A(H1N1)pdm09 viruses were similar to the 2018/2019 vaccine virus. Genetic data showed that A(H3N2) viruses mostly fell in clade 3C.2a (75%) and 90% of which were subclade 3C.2a1b. A lower proportion fell in clade 3C.3a (23%) and were antigenically distinct from the vaccine virus. All B/Victoria viruses belonged to clade 1A; 30% carried a double amino acid deletion in HA and were genetically and antigenically similar to the vaccine virus component, while 55% carried a triple amino acid deletion or no deletion in HA; these were antigenically distinct from each other and from the vaccine component. All B/Yamagata viruses belonged to clade 3 and were antigenically similar to the virus component in the quadrivalent vaccine for 2018/2019.ConclusionsA simultaneous circulation of genetically and antigenically diverse A(H3N2) and B/Victoria viruses was observed and represented a challenge to vaccine strain selection.     

Use of monoclonal anti-haemagglutinin antibodies for the “In vitro” selection of a sequential influenza virus antigenic variant

A sequential antigenic variant of the A/Texas/77 (H3N2) influenza virus was obtained in vitro using a monoclonal antibody against the haemagglutinin (HA) of the antigenic variant V18 previously selected in vitro from the parental Texas virus. The sequential antigenic variant, designated DV1, the V18 antigenic variant and the parental A/Texas/77 viruses were used to evaluate the frequency of anti-haemagglutinin antibodies in human sera in single radial haemolysis assays. Twenty six of 100 children's sera, which contained antibodies to the parental A/Texas/77 virus, failed to react with the V18 antigenic variant. A higher proportion of sera (42%) failed to react with the DV1 antigenic variant with alterations in two different antigenic determinants with respect to the parental virus. The results are discussed in relation to the mechanism of antigenic drift and to the in vivo reaction of antigenic variants selected in vitro.Corresponding author.