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.     

Studies on relationships between human and porcine influenza. 1. Serological evidence of infection in swine in Great Britain with an influenza A virus antigenically like human Hong Kong-68 virus

Serological evidence of infection of swine in Great Britain with an influenza A virus closely related to the human A/Hong Kong/68 (H3N2) variant was detected by a variety of serological tests. The Hong Kong/68 virus was first detected in man in Great Britain in August 1968 and was prevalent in the winters of 1968-69 and 1969-70. There was no evidence that swine had been infected with a Hong Kong/68-like virus before the appearance of the virus in man. The detection of virus-neutralizing antibody and high titres of neuraminidase-inhibiting antibody for Hong Kong/68 virus, and the production of precipitin lines corresponding to influenza A ribonucleoprotein and haemagglutinin and neuraminidase antigens of Hong Kong virus in immunodiffusion tests indicated that the swine sera contained antibody specific for the Hong Kong/68 virus. Evidence suggested that the infection of swine occurred in the early months of 1970. Clinical influenza among swine in Great Britain was not reported during the study period and there was no serological evidence of infection with “classical” swine influenzavirus strains.     

Experimental infection of human volunteers with a swine influenzavirus antigenically related to the human A-Hong Kong-68 virus

An influenzavirus of swine origin (swine/Taiwan/7310/70) antigenically closely related to the human A/Hong Kong/68 virus readily infected human volunteers. Those infected developed antihaemagglutinin and antineuraminidase antibodies to the human A/Hong Kong/68 virus as well as to the swine/Taiwan virus. The clinical reactions produced by the swine/Taiwan virus were, however, milder than those produced in volunteers infected with A/Hong Kong/68. In contrast, two other “classical” swine viruses (strains antigenically related to the prototype swine/Iowa/15/30 strain), immunologically distinct from the Hong Kong/68 virus, possessed low infectivity for man.     

Antigenic variation in current human type A influenza viruses: antigenic characteristics of the variants and their geographic distribution

Outbreaks of influenza due to the virus A/Hong Kong/1/68 (H3N2) began in 1968 and are still occurring. The haemagglutinin of this virus is different from that of the A/Singapore/1/57 virus (the “Asian” strain) but the neuraminidase antigens are the same. Between 1968 and 1971 only minor antigenic “drift” in the haemagglutinin was noted, but in recent months 2 isolates have been identified in which considerable “drift” has occurred in the haemagglutinin and in the neuraminidase antigens. One, A/Hong Kong/5/72 (H3N2), was first detected in outbreaks in Hong Kong between November 1971 and January 1972 and was predominant there and in Korea but did not become widely disseminated. The second strain, A/England/42/72 (H3N2), has been isolated in winter outbreaks in the southern hemisphere and now appears to be the predominant strain in the northern hemisphere. The characteristics of the strains are described.     

Characteristics of a swine recombinant influenza virus isolated in 1980: recombination between swine and the earliest Hong Kong (H3N2) viruses

A recombinant (H1N2, formerly Hsw1N2), A/swine/Ehime/1/80 was found to possess antigenic, biological and genomic characteristics different from those of a previous A/swine/Kanagawa/2/78 (H1N2) strain¹. Five monoclonal antibodies to A/NJ/8/76 differentiated the haemagglutinin molecules of the former virus from the latter, showing that these viruses differed at two-antigenic determinants at least. Immuno-double diffusion tests with antisera to the isolated neuraminidase and neuraminidase-inhibition tests with monoclonal antibodies to different H2N2 and H3N2 viruses revealed that A/swine/Ehime/1/80 strain contained a neuraminidase subunit very similar to that of late human Asian (H2N2) and the earliest Hong Kong (H3N2) viruses. RNA analysis by oligonucleotide mapping suggested that A/swine/Ehime/1/80 may be a recombinanat between A/swine/Shizuoka/1/78-like and A/Aichi/2/68 (H3N2)-like viruses. To determine further the gene constellation of this recombinant virus, DNA-RNA hybridizations were performed using DNA segments complementary for swine (H1N1) virus RNA and the entire RNA of three viruses. The molecular hybridization could define the genomic composition of the recombinant, indicating that only the neutraminidase gene of this virus is derived from the earliest Hong Kong (H3N2)-like virus and remaining seven genes are derived from swine (H1N1) virus.     

Antigenic variants of influenza A virus obtained in vitro.

The aim of this study was to investigate antigenic “drift” in the haemagglutinin and neuraminidase antigens of influenza A virus in vitro under immunological pressure. Variants of the “Asian” influenza strains A/England/12/64 (H2N2) and A/Tokyo/3/67 (H2N2) were isolated in the allantois-on-shell system in the presence of homologous postinfection ferret sera. For each of these two viruses three generations of variants were isolated and characterized. It was found that the successive antigenic variants of A/Eng/12/64 did not resemble A/Tokyo/3/67. Thus it is probable that the pathway of antigenic drift in vitro was not the same as that which occurred in nature during the evolution of A/Tokyo/3/67 from A/Eng/12/64. In addition, A/Tokyo/3/67, which was the last strain to be prevalent before the A/Hong Kong subtype appeared, underwent significant antigenic drift from “junior” to “senior” variants. This finding did not support the concept that, when antigenic drift occurs, resulting in the appearence of viruses with new haemagglutinin antigen subtypes, the previously prevalent strain has no capacity for further antigenic drift. The study did not result in the production of strains that were identifiable as “bridging” viruses between the H2 and H3 haemagglutinin subtypes. The present paper includes the first report of antigenic variation in the neuraminidase antigens of influenza A viruses occurring in vitro under immunological pressure.     

Serological diagnosis of human influenza infections by immunoprecipitin techniques

In immunodiffusion tests employing disrupted influenza virus (A2/Hong King/68, X-31 strain) as antigen, precipitin lines corresponding to three virus antigens can be distinguished—namely, the ribonucleoprotein, the haemagglutinin, and the neuraminidase. In the present study a comparison was made of such immunodiffusion tests and conventional diagnostic methods (complement-fixation and haemagglutination-inhibition tests) for serological diagnosis of A2/Hong Kong/68 infections in man. Precipitin tests in which the acquisition or reinforcement of precipitins for A2/Hong Kong/68 virus were detected, were found to be as sensitive as conventional methods for the serological diagnosis of influenza. In convalescent human sera precipitin lines corresponding to influenza A ribonucleoprotein were frequently detected, lines corresponding to A2 neuraminidase were less frequent, and those corresponding to haemagglutinin were least frequent. Precipitin tests had considerable advantages over other methods of serological diagnosis of influenza. They were rapid and simple to perform and were not susceptible to the effects of nonspecific reactions. In addition the antibody response to each of three antigens of the influenza virus could be detected in a single test system.     

Antigenic variation in current influenza A viruses: evidence for a high frequency of antigenic 'drift' for the Hong Kong virus

A new antigenic variant of the Hong Kong (H3N2) subtype of influenzavirus type A is described. The variant, A/Port Chalmers/1/73 (H3N2), was first isolated in Australasia in the autumn of 1973 and subsequently became the predominant influenza A variant in most areas of the world, replacing the previously prevalent strain A/England/42/72 (H3N2). The 1973 variant shows antigenic differences from former Hong Kong variants in both haemagglutinin and neuraminidase antigens. The application of immuno-double-diffusion tests and single-radial-diffusion tests in the antigenic analysis of new variants of the influenzavirus is also described. It is emphasized that since new variants of the Hong Kong virus have appeared in the successive years 1971, 1972, and 1973, the annual frequency of antigenic “drift” for the Hong Kong virus is higher than was recorded for the “Asian” influenzavirus (H2N2) in the first 5 years of the latter''s prevalence from 1957 to 1962, during which period little antigenic variation occurred.     

Characterization of the haemagglutinin and neuraminidase antigens of some recent avian type A influenzavirus isolates from Hong Kong

During 1969 type A influenzaviruses were isolated from three outbreaks of disease among domestic ducks in Hong Kong. The isolates were characterized in haemagglutination inhibition and neuraminidase inhibition tests with antisera to prototype avian and human influenza strains. A/duck/Hong Kong/46/69 and 120/69 contained haemagglutinin and neuraminidase antigens related to those of A/turkey/Wisconsin/66. The neuraminidases of these Hong Kong isolates, like that of turkey/Wisconsin/66, were antigenically closely related to those of human Asian influenzaviruses. A/duck/Hong Kong/826/69 contained haemagglutinin and neuraminidase antigens related to those of chicken/Scotland/59 and tern/South Africa/61, respectively. The duck influenza A isolates represent the first viruses of their antigenic variety to be isolated in South-East Asia. The possible epidemiological significance of the duck/Hong Kong/69 strains 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.     

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.     

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.     

Serological studies of influenza viruses in pigs in Great Britain 1991-2.

Samples from a sow serum bank representative of the pig population of Great Britain collected during 1991-2, were examined for antibodies to influenza A, B and C viruses, using viruses which had been isolated from a variety of hosts. For influenza A viruses there was evidence of the continued circulation of classical swine H1N1 virus (26%) seroprevalence), and human H3N2 viruses (39%) which are antigenically most closely-related to A/Port Chalmers/1/73 virus. In addition antibodies were detected to A/swine/England/201635/92 (8%), a strain of H3N2 virus which appears to have arisen by antigenic drift from conventional H3N2 swine strains. Specific antibodies (2%) were detected to an H1N1 virus (A/swine/England/195852/92) related most closely to avian H1N1 strains. In tests with human H1N1 and H3N2 viruses, excluding isolates from pigs, the highest seroprevalence was detected to the prevailing strains from the human population. Serological tests with avian H4 and H10, human H2, equine 1 and 2 influenza A viruses were all negative. Seven pigs seropositive by haemagglutination-inhibition, virus neutralization and immunoblotting assays for antibody to influenza B virus, were randomly distributed geographically suggesting that influenza B viruses may be transmitted to pigs but fail to spread. The seroprevalence to influenza C viruses was 9.9% indicating that these viruses are widespread in pigs. These results provide further evidence that the pig can be infected by a number of influenza viruses, some of which may have significance in the epidemiology of human influenza.     

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).     

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.     

Avian influenza A viruses of southern China and Hong Kong: ecological aspects and implications for man

Continuous surveillance of the influenza viruses isolated from domestic poultry from southern China and Hong Kong over more than 4 years resulted in the isolation of influenza viruses possessing 46 different combinations of haemagglutinin (H) and neuraminidase (N) subtypes. Of these, 43 were obtained from ducks from China. In all cases, infection appeared to be asymptomatic. The antigenic combination found most commonly in the viruses isolated was H4N6, which accounted for approximately one-quarter of the duck isolates, its occurrence being more frequent than expected from a statistical analysis of the observed frequencies of the haemagglutinin and neuraminidase genes among all the isolates. Some combinations of H and N occurred less frequently than expected or not at all. Influenza viruses tended to be isolated more frequently from ducks during the summer months in comparison with paramyxoviruses, which were more commonly encountered in the winter. Possible reasons for the great antigenic diversity of influenza A viruses in the poultry, especially the ducks, in the region are discussed together with the potential significance of these viruses to the emergence of human influenza pandemics.     

The use of transportable single-radial-diffusion immunoplates in seroepidemiological studies of influenza in the Gambia: The occurrence and persistence of antibody to influenza A/Hong Kong/68 (H3N2) virus in selected inhabitants of two rural villages

Seroepidemiological studies of influenza in the Gambia were made using transportable single-radial-diffusion immunoplates containing A/Hong Kong/68 (H3N2) virus as antigen. The frequency and durability of antibody so detected in selected residents of two Gambian villages (Manduar and Kafuta) are described. Transportable immunoplates were found to be an effective method for the serological surveillance of influenza and to be applicable in studies in remote areas where laboratory facilities may not be available. Results indicated that infection with influenza was widespread in Manduar residents on several occasions between 1968 and 1974 and that reinfection with A/Hong Kong/68 virus or its antigenic variants occurred frequently. Serum levels of antibodies to the haemagglutinin and neuraminidase antigens of the A/Hong Kong/68 virus often persisted for only a short time (mean half-life about 28 days), particularly after first infections. Antibody persistence increased following repeated reinfection. No precise explanation can be offered at present for the relatively short persistence of antibodies in Gambians. Possible reasons include genetic and environmental factors, depressed immunological reactivity associated with concurrent infection (notably parasitic diseases), and unusually high rates of synthesis and catabolism of immunoglobulins. The value of transportable immunoplates for serological surveys and for accurate assessment of antibody persistence is discussed.     

Antigenic comparisons of swine-influenza-like H1N1 isolates from pigs, birds and humans: an international collaborative study

The objective of this international collaborative study was to compare recent swine isolates of influenza viruses and determine whether significant antigenic differences among isolates from different areas of the world could be detected. H1N1 viruses isolated from pigs, birds and humans in 12 different countries were compared in haemagglutination-inhibition assays with post-infection ferret sera and monoclonal antibodies to H1N1 strains. Using A/NJ/8/76 as the reference strain, we found that recent swine isolates from Hong Kong, Italy, Japan, and the USA possess a haemagglutinin virtually indistinguishable from that of viruses typically associated with pigs, i.e., A/NJ/8/76. In contrast, recent swine isolates from several European countries (Belgium, Denmark, France, Federal Republic of Germany, and Spain) were distinguishable from A/NJ/8/76, as demonstrated by tests in the various laboratories. These studies suggest that the H1N1 viruses in pigs are antigenically heterogeneous and that the circulation of particular variants is associated with the geographical location of the animals. These results raise the question of whether these viruses originated from the same source, i.e., pigs, and have undergone antigenic drift or, alternatively, were introduced from other hosts, such as birds.     

Changes in the antibody status of a population following epidemic infection by influenza virus A2/Hong Kong/1/68

The haemagglutinin of influenza virus A2/Hong Kong/1/68 was shown to be markedly different from that of previously isolated A2 virus strains. No haemagglutination-inhibiting (HI) antibody to A2/Hong Kong/1/68 virus was detected in serum specimens collected in 1966 from persons aged 60 years or less. In contrast, HI antibody tests with 270 sera collected in 1968 indicated that 9·6% had demonstrable HI antibody at low titres, and 35·2% of 454 postepidemic (1969) sera had demonstrable HI antibody at relatively high titres. Most sera from persons aged 80 years and more collected in 1968 and 1969 had demonstrable HI antibody to influenza virus A2/Hong Kong/1/68. No HI antibody to the Hong Kong virus was detected in pre-epidemic sera from children aged 6 months to 3 years, whereas 32% of postepidemic sera had HI antibody. The acquisition of HI antibody to A2/Hong Kong/1/68 was not accompanied by an increase in the incidence or titres of HI antibody to heterotypic A2 influenza viruses. For sera from children aged 4-11 years, an increase of HI titre to heterotypic A2 influenza was found.     

Predominance of influenza A(H3N2) virus genetic subclade 3C.2a1 during an early 2016/17 influenza season in Europe – Contribution of surveillance data from World Health Organization (WHO) European Region to the WHO vaccine composition consultation for northern hemisphere 2017/18

During the European 2016/17 influenza season, A(H3N2) viruses have predominated and the majority clustered in genetic subclade 3C.2a1. Genetic analyses showed that circulating viruses have undergone considerable genetic diversification of the haemagglutinin gene from the current vaccine virus A/Hong Kong/4801/2014 (clade 3C.2a), but the antigenic data that is limited by the challenges with the antigenic characterisation of currently circulating A(H3N2) viruses, showed no clear evidence of antigenic change. The recommended A(H3N2) vaccine component for the northern hemisphere 2017/18 influenza season remained unchanged. However, early and mid-season vaccine effectiveness (VE) estimates were suggestive of reduced VE against A(H3N2) viruses.