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.     

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.     

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.     

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).     

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.     

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.     

Antigenic analysis of H2 influenza virus haemagglutinin with monoclonal antibodies

Antigenic analysis of human and avian H2 influenza virus was carried out with monoclonal antibodies to the HA molecules of H2 influenza viruses isolated in the early stage of an H2 pandemic. The study revealed antigenic differences between inhibitor sensitive (Japan⁺/57, RI⁺57) and inhibitor resistant strains (Japan^?/57, Ri^?/57). This indicates that the receptor-binding specificity of the haemagglutinin can markedly influence the antigenic analysis obtained with monoclonal antibodies in HI test. Minor antigenic differences (microheterogeneity) could be detected between different H2 influenza viruses isolated in 1957. Minor antigenic variation continued in the H2 viruses until 1961, but significant antigenic drift occurred in 1962 so that viruses isolated after that date reacted with few monoclonal antibodies. Analysis of avian H2 influenza viruses suggested antigenic differences between the different avian H2 haemagglutinin but no correlation between the year of isolation and the progressive antigenic drift similar to that seen in the human strains was found.     

Single-radial-haemolysis: a new method for the assay of antibody to influenza haemagglutinin: Applications for diagnosis and seroepidemiologic surveillance of influenza

A simple single-radial-haemolysis technique is described that permits the detection and assay of antibody to influenza virus haemagglutinin. The method depends on the passive haemolysis of virus-treated erythrocytes by antihaemagglutinin antibody and complement. Under the “standard” test conditions described antibody to the other surface antigen of the influenza virus (neuraminidase) or to the internal antigens of the virus (nucleoprotein and matrix protein) do not produce haemolysis. Because it requires only small amounts of crude virus antigen and is rapid and simple, the method appears to be of considerable value for large-scale seroepidemiologic studies of new influenza virus variants. Antihaemagglutinin antibody detected by single-radial-haemolysis appears to be relatively strain-specific; the technique may therefore be useful in the antigenic characterization of virus isolates.     

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.     

Avian influenza A viruses

The antigenic structure of eight strains of influenza A viruses of avian origin was investigated by haemagglutination inhibition, virus neutralization and strain-specific complement fixation. All strains could be distinguished from each other, but certain cross-reactions were observed allowing the establishment of four antigenic groupings, as follows: (1) classic fowl plague virus (“Dutch” strain), Turkey/England/63 (“Langham” strain) and virus N; (2) two strains isolated from ducks, one in Czechoslovakia in 1956 and one in England in 1962; (3) a third strain isolated from ducks in England in 1956; (4) the “Smith” strain (Chicken/Scotland/59) and the tern virus (Tern/South Africa/61).     

Cross-protection against a lethal influenza virus infection by DNA vaccine to neuraminidase

Cross-protection against a lethal influenza virus infection was examined in BALB/c mice immunized with plasmid DNAs encoding the neuraminidase (NA) from different subtype A viruses. Each NA-DNA was administered twice, 3 weeks apart, at the dose of 1 μg per mouse by particle-mediated DNA transfer to the epidermis (gene gun) or at a dose of 30 μg per mouse by electroporation into the muscle. Three weeks after the second vaccination, the mice were challenged with lethal doses of homologous or heterologous viruses and the ability of each NA-DNA to protect the mice from influenza was evaluated by determining the lung virus titers, body weight and survival rates. The H3N2 virus NA-DNA conferred cross-protection against lethal challenge with antigenic variants within the same subtype, but failed to provide protection against infection by a different subtype virus (H1N1). The degree of cross-protection against infection was related to titers of the cross-reacting antibodies. These results suggest that NA-DNA can be used as a vaccine component to provide effective protection against infection not only with homologous virus but also with drift viruses.     

An antigenic variant of the Hong Kong/68 influenza A2 virus

Both the haemagglutinin and the neuraminidase of influenza A2 virus have undergone progressive antigenic drift since this subtype first appeared in 1957.     

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.     

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.     

Antigenic and molecular heterogeneity in recent swine influenza A(H1N1) virus isolates with possible implications for vaccination policy

In order to explore the occurrence of antigenic drift in swine influenza A(H1N1) viruses and the match between epidemic and vaccine strains, 26 virus isolates from outbreaks of respiratory disease among finishing pigs in the Netherlands in the 1995/1996 season and reference strains from earlier outbreaks were examined using serological and molecular methods. In contrast to swine H3N2 viruses, no significant antigenic drift was observed in swine H1N1 viruses isolated from the late 1980s up to 1996 inclusive. However, a marked antigenic and genetic heterogeneity in haemagglutination inhibition tests and nucleotide sequence analyses was detected among the 26 recent swine H1N1 virus strains. Interestingly, the observed antigenic and molecular variants were not randomly distributed over the farms. This finding indicates independent introductions of different swine H1N1 virus variants at the various farms of the study and points to a marked difference between the epidemiologies of human and swine influenza viruses. The observed heterogeneity may hamper the control of swine influenza by vaccination and indicates that the efficacy of current swine influenza vaccines requires re-evaluation and that the antigenic reactivity of swine influenza viruses should be monitored on a regular basis.     

An investigation of antigenic drift of neuraminidases of influenza A (H1N1) viruses.

A newly developed lectin neuraminidase test (LNT) and a panel of mouse monoclonal and post-infection ferret antibodies have been used to analyse antigenic drift in N1 neuraminidases of influenza A viruses isolated between 1933 and 1957 and also between 1977 and 1980. Significant antigenic differences were detected among the ''early'' (1933-57) viruses since the NA of viruses isolated one year apart could be distinguished serologically. The NA of the ''re-emerged'' virus A/USSR/92/77 (H1N1) was antigenically related but not identical to influenza A viruses isolated in 1949 (A/Paris/49 (H1N1), A/Geneva/49 (H1N1) which thus predates the previously observed antigenic similarity of A/USSR/77 with A/FW/50 (H1N1) virus.     

Antigenic and genetic characterization of influenza viruses circulating in Bulgaria during the 2015/2016 season

Influenza virological surveillance is an essential tool for early detection of novel genetic variants of epidemiologic and clinical significance. The aim of this study was to determine the antigenic and molecular characteristics of influenza viruses circulating in Bulgaria during the 2015/2016 season. The season was characterized by dominant circulation of A(H1N1)pdm09 viruses, accounting for 66% of detected influenza viruses, followed by B/Victoria-lineage viruses (24%) and A(H3N2) viruses (10%). All sequenced influenza A(H1N1)pdm09, A(H3N2) and B/Victoria-lineage viruses belonged to the 6B.1, 3C.2a and 1A genetic groups, respectively. Amino acid analysis of 57 A(H1N1)pdm09 isolates revealed the presence of 16 changes in hemagglutinin (HA) compared to the vaccine virus, five of which occurred in four antigenic sites, together with 16 changes in neuraminidase (NA) and a number of substitutions in proteins MP, NP, NS and PB2. Despite the many amino acid substitutions, A(H1N1)pdm09 viruses remained antigenically closely related to A/California/7/2009 vaccine virus. Bulgarian A(H3N2) strains (subclade 3C.2a) showed changes at 11 HA positions four of which were located in antigenic sites A and B, together with 6 positions in NA, compared to the subclade 3C.3a vaccine virus. They contained unique HA1 substitutions N171K, S312R and HA2 substitutions I77V and G155E compared to Bulgarian 3C.2a viruses of the previous season. All 20 B/Victoria-lineage viruses sequenced harboured two substitutions in the antigenic 120-loop region of HA, and 5 changes in NA, compared to the B/Brisbane/60/2008 vaccine virus. The results of this study reaffirm the continuous genetic variability of circulating seasonal influenza viruses and the need for continued systematic antigenic and molecular surveillance.     

Comparison of influenza viruses isolated from man and from whales.

Four isolates of influenza virus strains from Moscow and Habarovsk that caused outbreaks of influenza in November and December 1977 in several cities of the USSR were studied and their haemagglutinins and neuraminidases were compared with those of other human and animal influenza viruses including A/whale/Pacific Ocean/76. In H1 tests these isolates, designated A/USSR/77, reacted with immune serum against A/FM/1/47 (H1N1) to the homologous titre, and with antiserum against A/whale/PO/19/76 virus to 1/8 of the homologous titre. In neuraminidase inhibition tests all A/USSR/77 isolates showed the presence of human N1 type neuraminidase, more closely related to A/sw/New Jersey/76 (Hsw1N1) than to A/FM/1/47 (H1N1) virus. The haemagglutinin of A/whale/Pacific Ocean/19/76 virus occupies an intermediate position between H0 and H1, but its neuraminidase is close to Nav2. The virus from whales multiplies better at low (28°C) and at high (40°C) temperatures than do the viruses of human origin that were tested.     

An investigation of antigenic drift of neuraminidases of influenza A (H1N1) viruses

A newly developed lectin neuraminidase test (LNT) and a panel of mouse monoclonal and post-infection ferret antibodies have been used to analyse antigenic drift in N1 neuraminidases of influenza A viruses isolated between 1933 and 1957 and also between 1977 and 1980. Significant antigenic differences were detected among the 'early' (1933-57) viruses since the NA of viruses isolated one year apart could be distinguished serologically. The NA of the 're-emerged' virus A/USSR/92/77 (H1N1) was antigenically related but not identical to influenza A viruses isolated in 1949 (A/Paris/49 (H1N1), A/Geneva/49 (H1N1) which thus predates the previously observed antigenic similarity of A/USSR/77 with A/FW/50 (H1N1) virus.